Humanized immunoglobulin loci

ABSTRACT

The present invention concerns methods and means to produce humanized antibodies from transgenic non-human animals. The invention specifically relates to novel immunoglobulin heavy and light chain constructs, recombination and transgenic vectors useful in making transgenic non-human animals expressing humanized antibodies, transgenic animals, and humanized immunoglobulin preparations.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/511,188 filed Jul. 29, 2009, which is a divisional application ofU.S. application Ser. No. 10/893,483 filed Jul. 15, 2004 (now U.S. Pat.No. 7,585,668) which claims priority under 35 U.S.C. Section 119(e) andthe benefit of U.S. Provisional Application Ser. No. 60/487,733 filedJul. 15, 2003, the entire disclosures of which are incorporated hereinby reference in their entireties.

In accordance with 37 CFR 1.821(e), we hereby expressly incorporateherein by reference, in its entirety, the last-filed (filed Apr. 4,2005) computer readable Sequence Listing, saved as “39691-0007A savedApr. 4, 2005.txt” date of creation Apr. 4, 2005, size 1,489 KB,submitted in U.S. application Ser. No. 10/893,483, filed Jul. 15, 2004.

FIELD OF THE INVENTION

The present invention concerns methods and means to produce humanizedantibodies from transgenic non-human animals. The invention specificallyrelates to novel immunoglobulin heavy and light chain constructs,recombination and transgenic vectors useful in making transgenicnon-human animals expressing humanized antibodies, transgenic animals,and humanized immunoglobulin preparations. The transgenic vectorscontain humanized immunoglobulin loci, which are capable of undergoinggene rearrangement, gene conversion and hypermutation in transgenicnon-human animals to produce diversified humanized antibodies, whileleaving the endogenous regulatory and antibody production machinery ofthe non-human animals essentially intact. The humanized antibodiesobtained have minimal immunogenicity to humans and are appropriate foruse in the therapeutic treatment of human subjects.

BACKGROUND OF THE INVENTION

Antibodies are an important class of pharmaceutical products that havebeen successfully used in the treatment of various human diseases andconditions, such as cancer, allergic diseases, prevention of transplantrejection and host-versus-graft disease.

A major problem of antibody preparations obtained from animals is theintrinsic immunogenicity of non-human immunoglobulins in human patients.In order to reduce the immunogenicity of non-human antibodies, it hasbeen shown that by fusing animal variable (V) region exons with humanconstant (C) region exons, a chimeric antibody gene can be obtained.

Humanized monoclonal antibodies have also been developed and are inclinical use. Humanized monoclonal antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in non-human animal, e.g.rodent, antibodies. Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature, 321: 522(1986); Riechmann et al., Nature, 332: 323 (1988); Verhoeyen et al.,Science, 239: 1534 (1988)), by substituting non-human animal, e.g.rodent, CDRs or CDR sequences for the corresponding sequences of a humanmonoclonal antibody.

It has been described that the homozygous deletion of the antibodyheavy-chain joining region (JH) gene in chimeric and germ-line mutantmice results in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993);Bruggemann et al., Year in Immunol., 7: 33 (1993). While this geneticengineering approach resulted in the expression of human immunoglobulinpolypeptides in genetically engineered mice, the level of humanimmunoglobulin expression is low. This may be due to species-specificregulatory elements in the immunoglobulin loci that are necessary forefficient expression of immunoglobulins. As demonstrated in transfectedcell lines, regulatory elements present in human immunoglobulin genesmay not function properly in non-human animals.

Indeed, several regulatory elements in immunoglobulin genes have beendescribed. Of particular importance are enhancers downstream (3′) ofheavy chain constant regions and intronic enhancers in light chaingenes. In addition, other, yet to be identified, control elements may bepresent in immunoglobulin genes. Studies in mice have shown that themembrane and cytoplasmic tail of the membrane form of immunoglobulinmolecules play an important role in expression levels of human-mousechimeric antibodies in the serum of mice homozygous for the human Cγ1gene. Therefore, for the expression of heterologous immunoglobulin genesin animals it is desirable to replace sequences that contain enhancerelements and exons encoding transmembrane (M1 exon) and cytoplasmic tail(M2 exon) with sequences that are normally found in the animal insimilar positions.

In addition to the issues raised by the potential immunogenicity of thenon-human antibodies, the use of monoclonal antibodies in general,whether chimeric, humanized or human, is further limited by the factthat devastating diseases, such as cancer and infections with virulentpathogens, are difficult to treat by attacking one target, due to theircomplexity, multifactorial etiology and adaptivity. Monoclonalantibodies directed against singularly defined targets fail when thosetargets change, evolve and mutate. Thus, malignancies may gainresistance to standard monoclonal antibody therapies. A solution to thisproblem is the use of polyclonal antibodies, which have the ability totarget and attack a plurality of evolving targets linked with complexdiseases. Polyclonal antibodies also have the ability to neutralizebacterial and viral toxins, and direct immune responses to kill andeliminate pathogens.

Accordingly, there is a great clinical need for a new approach suitablefor the large-scale production of high-titer, high-affinity, humanizedpoly- and monoclonal antibodies.

The introduction of human immunoglobulin genes into the genome of miceresulted in expression of a diversified human antibody repertoire ingenetically engineered mice. In both mice and humans, primary antibodydiversity is generated by gene rearrangement. This process results inthe generation of many different recombined V(D)J segments encoding alarge number of antibody molecules with different antigen binding sites.However, in other animals, like rabbits, pigs, cows and birds, primaryantibody diversity is generated by substantially different mechanisms,namely templated mutations or gene conversion and non-templatedmutations or hypermutation. For example, it is well established that inrabbit and chicken, VDJ rearrangement is very limited (almost 90% ofimmunoglobulin is generated with the 3′ proximal VH1 element) andantibody diversity is generated by gene conversion and hypermutation. Incontrast, mouse and human gene conversion occurs very rarely, if at all.Therefore, it is expected that in animals that diversify their primaryantibody repertoire by gene conversion and hypermutation a geneticengineering approach based on gene rearrangement will result in animalswith low antibody titers and limited antibody diversity. Thus, thegenetic engineering of large animals for the production ofnon-immunogenic antibody preparations for human therapy requiresalternative genetic engineering strategies.

The production of humanized antibodies in transgenic non-human animalsis described in PCT Publication No. WO 02/12437, published on Feb. 14,2002, the disclosure of which is hereby expressly incorporated byreference in its entirety. WO 02/12437 describes genetically engineerednon-human animals containing one or more humanized immunoglobulin lociwhich are capable of undergoing gene rearrangement and gene conversionin transgenic non-human animals, including animals in which antibodydiversity is primarily generated by gene conversion to producediversified humanized antibodies. The humanized antibodies obtained haveminimal immunogenicity to humans and are appropriate for use in thetherapeutic treatment of human subjects. It further describes novelnucleotide sequences from the 5′ and 3′ flanking regions ofimmunoglobulin heavy chain constant region segments of various non-humanmammalians, such as chickens, cows, sheep, and rabbits. Recombinantvectors in which human immunoglobulin heavy chain gene segments areflanked by sequences homologous to such 5′ and 3′ sequences are shown tobe useful for replacing an immunoglobulin heavy chain gene segment of anon-human animal with the corresponding human immunoglobulin heavy chaingene segment.

SUMMARY OF THE INVENTION

In one aspect, the present invention concerns an isolated nucleic acidmolecule comprising a human immunoglobulin gene segment, flanked bynucleotide sequences, wherein the flanking sequences are identical ordifferent, and comprise at least about 20 contiguous nucleotides of aspacer sequence from an immunoglobulin heavy or light chain gene of ananimal generating antibody diversity primarily by gene conversion and/orhypermutation, or from a consensus sequence of two or more of the spacersequences.

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising a human immunoglobulin heavy or light chain constantregion (C) gene segment, flanked by nucleotide sequences, wherein theflanking sequences are identical or different, and comprise at leastabout 20 contiguous nucleotides of a spacer sequence from animmunoglobulin heavy or light chain gene of a non-primate animal, orfrom a consensus sequence of two or more of the spacer sequences.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a human immunoglobulin heavy or light chain genesegment, flanked by nucleotide sequences, wherein the flanking sequencesare identical or different, and comprise at least about 20 contiguousnucleotides of a spacer sequence selected from the group consisting ofSEQ ID NOS: 1 to 185 (Table 1), or from a consensus sequence of two ormore of the spacer sequences.

In one embodiment, the flanking sequences comprise at least about 50contiguous nucleotides of a spacer sequence.

In another embodiment, the human immunoglobulin gene segment is a heavychain V, D, or J segment, where the V fragment may, for example be amember of the VH3, VH1, VH5, or VH4 family.

In a further embodiment, the human immunoglobulin gene segment is alight chain V or J segment, where the V segment may, for example be a κlight chain gene segment, such as Vκ1, Vκ3, or Vκ4, or a λ light chainsegment, e.g. Vλ1, Vλ2 or Vλ3.

In a further embodiment, the non-primate animal which generates antibodydiversity primarily by gene conversion and/or somatic hypermutation is,for example, rabbit, pig, bird, e.g. chicken, turkey, duck, or goose,sheep, goat, cow, horse or donkey, however, other non-primate animals,e.g. rodents are also specifically included within the scope of theinvention.

In another aspect, the invention concerns a recombination vectorcomprising any of the foregoing nucleic acid molecules.

In yet another aspect, the invention concerns a transgenic vectorcomprising a humanized immunoglobulin (Ig) locus, wherein the humanizedIg locus is derived from an Ig locus or a portion of an Ig locus of anon-human animal, comprising multiple Ig segments wherein

(a) at least one of the gene segments is a human Ig gene segment flankedby nucleotide sequences comprising at least about 20 contiguousnucleotides from a spacer sequence, or from a consensus sequence or twoor more of such spacer sequences;

(b) the gene segments are juxtaposed in an unrearranged, partiallyrearranged or fully rearranged configuration, and

(c) the humanized Ig locus is capable of undergoing gene rearrangement,if necessary, as well as gene conversion and/or hypermutation, if thenon-human animal is a gene converting animal, and producing a repertoireof humanized immunoglobulins in the non-human animal.

In a further embodiment, the humanized Ig heavy chain locus present inthe transgenic vector comprises about 5 to 100 V gene segments, with atleast one human V gene segment. In a specific embodiment, the humanizedIg heavy chain locus comprises more than one human V gene segments.

In another embodiment, the humanized Ig heavy chain locus present in thetransgenic vector comprises about 5 to 25 D gene segments, In a specificembodiment, the humanized Ig heavy chain locus comprises one or severalhuman D gene segments.

In yet another embodiment, the humanized Ig heavy chain locus present inthe transgenic vector comprises about 1 to 10 J gene segments, with atleast one human J gene segment. In a specific embodiment, the humanizedIg heavy chain locus comprises more than one human J gene segments.

In another embodiment, the humanized Ig heavy chain locus present in thetransgenic vector comprises about 1-25 C region segments, with at leastone human C region segment. In a specific embodiment, the humanized Igheavy chain locus present in the transgenic vector comprises more thanone human C gene segment.

In a still further embodiment, the humanized Ig locus present in thetransgenic vector is a light chain locus of a non-human animal, and itcomprises at least one V gene segment, at least one J gene segment andat least one constant (C) region gene segment, where at least one genesegment is selected from the group of human light chain V and J segmentsand human light chain C region segments. In a specific embodiment, theconstant region gene segment is a human light chain constant region genesegment, which can, for example, be a Cλ or Cκ gene segment. In anotherembodiment, the humanized Ig light chain locus comprises two or moresegments selected from human V and J segments and human C regionsegments. In a further embodiment, the humanized Ig light chain locuscomprises at least one human V segment, at least one human J segment,and at least one human C region segment.

In a further embodiment, the humanized Ig light chain locus present inthe transgenic vector comprises about 5-100 V gene segments, with atleast one human V gene segment, wherein the human V gene segment isplaced downstream to the 5-100 V gene segments of the non-human animal.In a specific embodiment, the human V gene segment is placed immediately5′ to a J gene segment in a rearranged configuration. In anotherembodiment, the humanized Ig light chain locus present in the transgenicvector comprises more than one human V gene segment.

In a still further embodiment, the humanized Ig light chain locuspresent in the transgenic vector comprises about 1-10 J gene segments,with at least one human J gene segment. In a specific embodiment, thehumanized Ig light chain locus present in the transgenic vectorcomprises more than one human J gene segment.

In another embodiment, the humanized Ig light chain locus present in thetransgenic vector comprises about 1-25 C region segments, with at leastone human C region segment. In a specific embodiment, the humanized Iglight chain locus present in the transgenic vector comprises more thanone human C gene segment.

In a still further embodiment, the humanized Ig locus present in thetransgenic vector is a light chain locus of a non-human animal, and itcomprises at least one V gene segment, at least one J gene segment andat least one constant (C) region gene segment, where at least one genesegment is selected from the group of human light chain V and J segmentsand human light chain C region segments. In a specific embodiment, theconstant region gene segment is a human light chain constant region genesegment, which can, for example, be a Cλ or Cκ gene segment. In anotherembodiment, the humanized Ig light chain locus comprises two or moresegments selected from human V and J segments and human C regionsegments. In a further embodiment, the humanized Ig light chain locuscomprises at least one human V segment, at least one human J segment,and at least one human C region segment.

In a different aspect, the invention concerns a nucleic acid moleculecomprising two or more units consisting of, from 5′ to 3′ direction, a5′ nucleotide sequence, a human immunoglobulin sequence, and a 3′nucleotide sequence, wherein the 5′ and 3′ nucleotide sequences areidentical or different, and comprise at least about 20 contiguousnucleotides from a spacer sequence separating the coding regions in anon-primate animal immunoglobulin heavy or light chain gene, or from aconsensus sequence of two or more of the spacer sequences. In a specificembodiment, the spacer sequences are selected from within SEQ ID NOS: 1to 185 (Table 1). In another particular embodiment, the 5′ and/or 3′nucleotide sequences in all repetitive units of the nucleic acidmolecule are identical. In another particular embodiment, the repetitiveunits of the nucleic acid molecule comprise at least two different 5′and/or 3′ sequences. In a further embodiment, the 5′ and 3′ nucleotidesequence are different from each other, but all 5′ and all 3′ nucleotidesequences are identical.

In a further aspect, the invention concerns a method for making atransgenic vector comprising a humanized immunoglobulin (Ig) locuscapable of producing a functional repertoire of humanized antibodies ina non-human animal, comprising:

(a) obtaining a DNA fragment comprising an Ig locus or a portion thereoffrom the non-human animal which comprises at least one V gene segment,at least one J gene segment and at least one constant region genesegment; and

(b) integrating at least one human Ig gene segment into the DNA fragmentof step (a) to produce a humanized Ig locus, wherein the human Ig genesegment flanked by nucleotide sequences comprising at least about 20contiguous nucleotides from a spacer sequence separating the codingregions in a non-primate animal immunoglobulin heavy or light chaingene, or from a consensus sequence or two or more of such spacersequences; wherein (i) the gene segments are juxtaposed in anunrearranged, partially rearranged or fully rearranged configuration,and (ii) the humanized Ig locus is capable of undergoing generearrangement, if necessary, and producing a repertoire of humanizedimmunoglobulins in the non-human animal.

The humanized Ig locus can be a humanized Ig heavy chain or light chainlocus. In the case of a humanized Ig heavy chain locus the DNA fragmentobtained in step (a) additionally comprises at least one D gene segment.

In another aspect, the invention concerns a transgenic animal comprisinga humanized immunoglobulin locus described above, and methods for makingsuch transgenic animals. In one embodiment, the transgenic animalcomprises both a humanized immunoglobulin heavy chain locus and ahumanized immunoglobulin light chain locus. In another embodiment, onlyone of the heavy and light chain loci present in the transgenic animalis humanized. In another embodiment, all of the V, D, J and C regions ofat least one of the animal's immunoglobulin loci are humanized. In yetanother embodiment, all of the V, D, J, and C region of the transgenicanimals endogenous immunoglobulin loci are humanized.

In a further aspect, the invention concerns a B cell from the transgenicanimals produced in accordance with the present invention.

In a still further aspect, the invention concerns a humanizedimmunoglobulin produced using a transgenic animal of the presentinvention, and an antibody preparation or a pharmaceutical compositioncomprising the humanized immunoglobulin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the rabbit immunoglobulin gene heavychain locus.

FIGS. 2-4 show a comparison of rabbit heavy chain spacer sequences.

FIG. 5 is a schematic depiction of the rabbit immunoglobulin light chainlocus.

FIG. 6 illustrates the building of an immunoglobulin gene V locus usinghuman V_(H) and rabbit spacer elements.

FIGS. 7 (a) and (b): Insertion of two cassettes by homologousrecombination using the redϵβγ-system. FIG. 7(a) shows that the uppercassette contains two restriction sites (FseI and AscI) flanking agentamycin cassette. FIG. 7(b) shows that the lower cassette contains aninverted (i) and mutated (71) loxP-site, a FRT-site and aMluI-restriction site. After modification the BAC is digested with FseIand AscI.

FIG. 8 shows a humanized rabbit light chain locus (rLC3-B) based on therabbit K1 light chain locus. Rabbit Cκ1 was replaced with human Cκ. Ahuman rearranged human VκJκ was inserted. The synthetic human VκJκshares more than 80% sequence homology with rabbit Vκ elements

FIG. 9a shows the sequence of a BAC clone comprising a chicken lightchain genomic locus whose nucleotide sequence is shown in FIG. 9b (SEQID NO: 186).

FIGS. 10a and 10b illustrate an outline showing the construction of ahumanized immunoglobulin locus using chicken immunoglobulin spacersequences and human V elements.

FIGS. 11a and 11b illustrate an outline showing the construction of ahumanized immunoglobulin locus using mouse or rabbit immunoglobulinspacer sequences and human V elements.

FIGS. 12a to 12c show a humanized light chain locus. A syntheticsequence (FIG. 12a, Unit 1, 12,235 bp, SEQ ID NO: 187) containing 17human V pseudogenes and 18 chicken spacer sequences is shown in (a). Asecond synthetic sequence (FIG. 12b, Unit 2, 13,283 bp, SEQ ID NO: 188)containing a functional rearranged human VkJk gene fragment, 11 human Vpseudogenes, 12 chicken spacer sequences and 2 introns is shown in (b).Units 1 and 2 were combined with a fragment derived from BAC 179L1containing human Ck and rabbit intron and spacer sequences (FIG. 12c).

FIG. 13a-e show a humanized heavy chain locus. Four synthetic DNAfragments (Unit 1-4, FIG. 13a-d, SEQ ID NOS: 189, 190, 191, 192)consisting of human VH3 gene fragments and rabbit spacer and intronsequences were combined with parts of BAC 219D23, 27N5 and Fos15B asshown in (FIG. 13e).

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by covalent disulfide bond(s), while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains (Clothia et al., J. Mol.Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A.82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, connected by three CDRs. TheCDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” is used to refer to an antibody moleculesynthesized by a single clone of B cells.

The term “polyclonal antibody” is used to refer to a population ofantibody molecules synthesized by many clones of B cells. In a specificembodiment, polyclonal antibodies recognize several epitopes.

The terms “a humanized antibody” and “a humanized immunoglobulin”, asused herein, mean an immunoglobulin molecule comprising at least aportion of a human immunoglobulin polypeptide sequence (or a polypeptidesequence encoded by a human immunoglobulin gene segment). The humanizedimmunoglobulin molecules of the present invention can be isolated from atransgenic non-human animal engineered to produce humanizedimmunoglobulin molecules. Such humanized immunoglobulin molecules areless immunogenic to primates, especially humans, relative tonon-humanized immunoglobulin molecules prepared from the animal orprepared from cells derived from the animal.

The term “non-human animal” as used herein includes, but is not limitedto mammals, and includes, for example, non-human primates, rabbits,pigs, birds (e.g., chickens, turkeys, ducks, geese and the like), sheep,goats, cows, horses, and rodents (e.g. mice and rats). Preferrednon-human animals are those animals which create antibody diversitysubstantially by gene conversion and/or somatic hypermutation, e.g.,rabbit, pigs, birds (e.g., chicken, turkey, duck, goose and the like),sheep, goat, and cow. Particularly preferred non-human animals arerabbit and chicken.

The term “non-primate animal” as used herein includes, but is notlimited to, mammals other than primates, including those listed above.

The phrase “animals which create antibody diversity substantially bygene conversion and/or hypermutation” is used to refer to animals inwhich the predominant mechanism of antibody diversification is geneconversion and/or hypermutation as opposed to gene rearrangement.

The term “Ig gene segment” as used herein refers to segments of DNAencoding various portions of an Ig molecule, which are present in thegermline of animals and humans, and which are brought together in Bcells to form rearranged Ig genes. Thus, Ig gene segments as used hereininclude V gene segments, D gene segments, J gene segments and C regiongene segments.

The term “human Ig gene segment” as used herein includes both naturallyoccurring sequences of a human Ig gene segment, degenerate forms ofnaturally occurring sequences of a human Ig gene segment, as well assynthetic sequences that encode a polypeptide sequence substantiallyidentical to the polypeptide encoded by a naturally occurring sequenceof a human Ig gene segment. By “substantially” is meant that the degreeof amino acid sequence identity is at least about 85%-95%. In aparticular embodiment, the human Ig gene segment renders theimmunoglobulin molecule non-immunogenic in humans.

A specific humanized immunoglobulin molecule of the present inventioncontains at least a portion of a human heavy or light chain variableregion polypeptide sequence. Another specific immunoglobulin moleculecontains at least a portion of a human heavy or light chain variableregion polypeptide sequence, and at least a portion of a human constantdomain polypeptide sequence.

By “a preparation of humanized antibodies” or “a humanized antibodypreparation” is meant an isolated antibody product or a purifiedantibody product prepared from a transgenic non-human animal (e.g.,serum, milk, or egg yolk of the animal) or from cells derived from atransgenic non-human animal (e.g., a B-cell or a hybridoma cell).

A humanized antibody preparation can be a preparation of polyclonalantibodies, which includes a repertoire of humanized immunoglobulinmolecules. A humanized antibody preparation can also be a preparation ofa monoclonal antibody.

The terms “antibody diversity” and “antibody repertoire” are usedinterchangeably, and refer to the total of all antibody specificitiesthat an organism is capable of expressing.

The term “spacer sequence” is used herein to refer to any non-codingnucleotide sequence present in an immunoglobulin heavy or light chaingene. Thus, the term specifically includes intron sequences and anyother non-coding sequences separating the coding regions within the V,D, J segments and C region segments in an immunoglobulin heavy chaingene, intron sequences and any other non-coding sequences separating thecoding regions within the V and J segments an C region segments in animmunoglobulin light chain gene, as well as non-coding sequence flankingregulatory elements, such as enhancers, in an immunoglobulin heavy orlight chain gene. In addition, non-coding sequences between exonsencoding parts of a membrane-spanning helix and heavy and light chainenhancers are specifically included.

An Ig locus having the capacity to undergo gene rearrangement and geneconversion is also referred to herein as a “functional” Ig locus, andthe antibodies with a diversity generated by a functional Ig locus arealso referred to herein as “functional” antibodies or a “functional”repertoire of antibodies.

B. Relevant Literature

Regulatory elements in immunoglobulin genes have been described byBradley et al. (1999), Transcriptional enhancers and the evolution ofthe IgH locus; Lauster, R. et al., Embo J 12: 4615-23 (1993); Volgina etal., J Immunol 165:6400 (2000); Hole et al., J Immunol 146:4377 (1991).

Antibody diversification by gene conversion in chicken and rabbit hasbeen described by Bucchini et al., Nature 326: 409-11 (1987); Knight etal., Advances in Immunology 56: 179-218 (1994); Langman et al., ResImmunol 144: 422-46 (1993). The generation of mice expressinghuman-mouse chimeric antibodies has been described by Pluschke et al.,Journal of Immunological Methods 215: 27-37 (1998). The generation ofmice expressing human-mouse chimeric antibodies with mouse derivedmembrane and cytoplamic tails has been described by Zou et al., Science262: 1271-1274 (1993); Zou et al. Curr Biol 4: 1099-1103. The generationof mice expressing human immunoglobulin polypeptides has been describedby Bruggemann et al. Curr Opin Biotechnol 8(4): 455-8 (1997); Lonberg etal. Int Rev Immunol 13(1):65-93 (1995); Neuberger et al., Nature 338:350-2 (1989). Generation of transgenic mice using a BAC clone has beendescribed by Yang et al., Nat Biotechnol 15: 859-65 (1997). Thegeneration of cows expressing human antibodies has been described byKuroiwa et al., Nature Biotech 20(9): 889-894 (2002).

The generation of transgenic rabbits has been described by Fan, J. etal., Pathol Int 49: 583-94 (1999); Brem et al., Mol Reprod Dev 44: 56-62(1996). Rabbits with impaired immunoglobulin expression have beendescribed by McCartney-Francis et al., Mol Immunol 24: 357-64 (1987);Allegrucci, et al., Eur J Immunol 21: 411-7 (1991).

The production of transgenic chicken has been described by Sherman etal., Nature Biotech 16:1050-1053 (1998); Etches et al., Methods inMolecular Biology 62: 433-450; Pain et al., Cells Tissues Organs165(3-4): 212-9 (1999); Sang, H., “Transgenic chickens—methods andpotential applications”, Trends Biotechnol 12:415 (1994); and inWO2004003157, “Gene regulation in transgenic animals using a transposonbased vector”; and in WO 200075300, “Introducing a nucleic acid into anavian genome, useful for transfecting avian blastodermal cells forproducing transgenic avian animals with the desired genes, by directlyintroducing the nucleic acid into the germinal disc of the egg”.

A gammaglobulinemic chicken have been described by Frommel et al., JImmunol 105(1): 1-6 (1970); Benedict et al., Adv Exp Med Biol 1977;88(2): 197-205.

The cloning of animals from cells has been described by T. Wakayama etal., Nature 1998; 394:369-374; J. B. Cibelli et al., Science280:1256-1258 (1998); J. B. Cibelli et al., Nature Biotechnology 1998;16:642-646; A. E. Schnieke et al., Science 278: 2130-2133 (1997); K. H.Campbell et al., Nature 380: 64-66 (1996), Kuroiwa et al., NatureGenetics 2004, Jun. 6. Nuclear transfer cloning of rabbits has beendescribed by Stice et al., Biology of Reproduction 39: 657-664 (1988),and Challah-Jacques et al., Cloning and Stem Cells 8(4):295-299 (2003).

Production of antibodies from transgenic animals is described in U.S.Pat. No. 5,814,318, No. 5,545,807 and No. 5,570,429. Homologousrecombination for chimeric mammalian hosts is exemplified in U.S. Pat.No. 5,416,260. A method for introducing DNA into an embryo is describedin U.S. Pat. No. 5,567,607. Maintenance and expansion of embryonic stemcells is described in U.S. Pat. No. 5,453,357.

The mechanisms involved in the diversification of the antibodyrepertoire in pigs, sheep and cows are reviewed in Butler, J. E. (1998),“Immunoglobulin diversity, B-cell and antibody repertoire development inlarge farm animals”, Rev Sci Tech 17:43. Antibody diversification insheep is described in Reynaud, C. A., C. Garcia, W. R. Hein, and J. C.Weill (1995), “Hypermutation generating the sheep immunoglobulinrepertoire is an antigen-independent process”, Cell 80:115; and Dufour,V., S. Malinge, and F. Nau. (1996), “The sheep Ig variable regionrepertoire consists of a single VH family,” J Immunol 156:2163.

C. Detailed Description

Immunoglobulin heavy and light chain genes comprise several segmentsencoded by individual genes and separated by intron sequences. Thusgenes for the human immunoglobulin heavy chain are found on chromosome14. The variable region of the heavy chain (VH) comprises three genesegments: V, D and J segments, followed by multiple genes coding for theC region. The V region is separated from the C region by a large spacer,and the individual genes encoding the V D and J segments are alsoseparated by spacers.

There are two types of immunoglobulin light chains: κ and λ. Genes forthe human κ light chain are found on chromosome 2 and genes for thehuman λ light chain are found on chromosome 22. The variable region ofantibody light chains includes a V segment and a J segment, encoded byseparate gene segments. In the germline configuration of the κ lightchain gene, there are approximately 100-200 V region genes in lineararrangement, each gene having its own leader sequence, followed byapproximately 5 J gene segments, and C region gene segment. All Vregions are separated by introns, and there are introns separating theV, J and C region gene segments as well.

The immune system's capacity to protect against infection rests in agenetic machinery specialized to create a diverse repertoire ofantibodies. Antibody-coding genes in B cells are assembled in a mannerthat allows to countless combinations of binding sites in the variable(V) region. It is estimated that more than 10¹² possible bindingstructures arise from such mechanisms. In all animals, including humans,the antibody-making process begins by recombining variable (V),diversity (D) and joining (J) segments of the immunoglobulin (Ig) locus.Following this step, depending on the animal species, two generalmechanisms are used to produce the diverse binding structures ofantibodies.

In some animals, such as human and mouse, there are multiple copies ofV, D and J gene segments on the immunoglobulin heavy chain locus, andmultiple copies of V and J gene segments on the immunoglobulin lightchain locus. Antibody diversity in these animals is generated primarilyby gene rearrangement, i.e., different combinations of gene segments toform rearranged heavy chain variable region and light chain variableregion. In other animals (e.g., rabbit, birds, e.g., chicken, goose, andduck, sheep, goat, and cow), however, gene rearrangement plays a smallerrole in the generation of antibody diversity. For example, in rabbit,only a very limited number of the V gene segments, most often the V genesegments at the 3′ end of the V-region, is used in gene rearrangement toform a contiguous VDJ segment. In chicken, only one V gene segment (theone adjacent to the D region, or “the 3′ proximal V gene segment”), oneD segment and one J segment are used in the heavy chain rearrangement;and only one V gene segment (the 3′ proximal V segment) and one Jsegment are used in the light chain rearrangement. Thus, in theseanimals, there is little diversity among initially rearranged variableregion sequences resulting from junctional diversification. Furtherdiversification of the rearranged Ig genes is achieved by geneconversion, a process in which short sequences derived from the upstreamV gene segments replace short sequences within the V gene segment in therearranged Ig gene. Additional diversification of antibody sequences maybe generated by hypermutation.

Immunoglobulins (antibodies) belong into five classes (IgG, IgM, IgA,IgE, and IgD, each with different biological roles in immune defense.The most abundant in the blood and potent in response to infection isthe IgG class. Within the human IgG class, there are four sub-classes(IgG1, IgG2, IgG3 and IgG4 isotypes) determined by the structure of theheavy chain constant regions that comprise the Fc domain. The F(ab)domains of antibodies bind to specific sequences (epitopes) on antigens,while the Fc domain of antibodies recruits and activates othercomponents of the immune system in order to eliminate the antigens.

Antibodies have been used successfully as therapeutics since the 1890swhen it was found that polyclonal antiserum taken from animals couldtreat life-threatening infections in humans. A significant advance inantibody research occurred with the development of methods for therecombinant production of antibodies, followed by the development ofantibody humanization techniques and method for making fully humanmonoclonal antibodies in non-human animals.

As a result, chimeric, humanized and human monoclonal antibodies haverecently emerged as an important class of pharmaceutical products. Whilemonoclonal antibody-based drugs are very effective in treating diseaseswhen blocking a particular target (e.g. receptor or ligand) certaindevastating diseases, such as cancer and infections with virulentpathogens, may be difficult to treat due to their complexity,multifactoral etiology and adaptivity. Monoclonal antibodies addresssingularly defined targets that change, evolve and mutate during thespread of diseases throughout a population or within an individual. Suchadaptive evolution is the bane of mono-specific drugs (e.g. monoclonalantibodies), which are quickly circumvented by resistant strains.Examples abound of bacterial and viral resistance to high-potencyantibiotics, and malignant cancers that develop resistance to standardanticancer drugs, such as monoclonal antibody therapies.

In contrast, polyclonal antibodies have the ability to bind andeliminate a plurality of evolving targets linked with complex diseases.By binding multiple antigens, polyclonal antibodies saturate the targetand retain activity even in the event of antigen mutation. Followingthis, through a cascade of signals, polyclonal antibodies induce apotent immune response to eliminate the target antigen, pathogen orcell. These properties make polyclonal antibodies ideal for treatinginfectious diseases and cancer.

So far, the use of polyclonal antibodies has been severely limited byeither supply problems or unwanted reactions to non-human proteins.

The present invention provides a new humanization approach, based onselective humanization the immunoglobulin-coding elements of theimmunoglobulin (Ig) translocus. The creation of such human-animaltranslocus allows for the creation of transgenic animals that expressdiversified, high-affinity humanized (polyclonal) antibodies in highyields.

As a first step, the genomic loci for non-human, including non-primate,immunoglobulin heavy and light chains are identified and sequenced. Forexample, as part of the present invention, genomic sequences for rabbitand chicken immunoglobulin heavy and light chains were determined, andare shown in FIGS. 1, 5, and 9.

Analysis of the rabbit Ig heavy chain genomic locus has shown that theimmunoglobulin heavy chain variable region (Vh) contains numerous genes,including functional genes and non-functional pseudogenes. Alignment of18 Vh genes has revealed a high degree (80-90%) sequence identity amongrabbit heavy chain variable region gene sequences (Vh1-Vh18). The rabbitheavy chain variable region genes have been found to share highesthomology with the Vh3 group of the human heavy chain variable regiongenes. Specifically, sequence comparison of the rabbit Vh1-a2 gene withthe human Vh3-23 sequences has revealed 72.8% sequence identity.

In addition, the non-coding (e.g. intron) sequences separating therabbit heavy chain variable region gene sequences were analyzed. FIGS.2-4 show a comparison of rabbit heavy chain intron sequences. It hasbeen found that such intron sequences fall into two groups, and arehighly conserved. Especially members of the Group 1 introns show asurprisingly high (80-90%) sequence identity.

Similar findings were made by analysis of rabbit immunoglobulin lightchain variable region genomic sequences. In particular, analysis of therabbit immunoglobulin light chain locus has shown that the light chainvariable region (V1) region contains numerous gene segments, which showa high degree (80-90% sequence identity). It has further been found thatthe rabbit light chain variable region (Vκ) exhibits high homology withthe Vκ1 group of the human light chain variable region gene sequences.Most Vκ sequences have been found to be functional and highly conserved.Unlike in the rabbit heavy chain variable region genes, in the rabbitlight chain variable region genes the intron sequences have been foundto be heterogeneous.

Similar studies with chicken immunoglobulin heavy and light chaingenomic sequences provide analogous results.

In one aspect, the present invention provides spacer sequences, whichseparate the coding regions in a non-primate animal heavy or light chaingene. In one embodiment, the present invention provides spacer sequencesfrom the heavy and light chain genes of animals which create antibodydiversity substantially by gene conversion, including, for example,rabbit and chicken. Such spacer sequences are then used to flank humanimmunoglobulin heavy or light chain gene segments used in the process ofcreating a humanized immunoglobulin locus.

The spacer sequences typically comprise at least about 20 nucleotides,or at least about 30 nucleotides, or at least about 40 nucleotides, orat least about 50 nucleotides, and typically are between about 20 andabout 10000 nucleotides in length. The spacer sequences may contain acontiguous stretch of nucleotides of appropriate length from a naturallyoccurring intron sequence in a non-human (e.g. non-primate) animal, ormay include an artificial sequence, which may, for example, be aconsensus sequence of two or more naturally occurring intron sequences.

The spacer sequences may comprise at least about 20 (30, 40, 50, etc. upto 1000 in 10-nucleotide increments) contiguous nucleotides from asequence selected from the group consisting of SEQ ID NOS 1 to 185(Table 1), or from a consensus sequence of two or more of suchsequences. It is possible, but not necessary, to separate human heavy orlight chain sequences (e.g. V, D, J, C region sequences) used forhumanization by spacer sequences that separate the corresponding regionswithin the genomic sequence of the non-human (non-primate) animal theimmunoglobulin of which is humanized.

In general, the humanization of an immunoglobulin (Ig) locus in anon-human animal involves the integration of one or more human Ig genesegments into the animal's genome to create humanized immunoglobulinloci. Thus, creation of a humanized Ig heavy chain locus involves theintegration of one or more V and/or D and/or J segments, and/or C regionsegments into the animal's genome. Similarly, the creation of ahumanized Ig light chain locus involves the integration of one or more Vand/or J segments, and/or C region segments into the animal's genome.

Depending upon the approach used, the human Ig gene segment(s) can beintegrated at the chromosomal location where the endogenous Ig locus ofthe animal ordinarily resides, or at a different locus of the animal.Regardless of the chromosomal location, the humanized Ig locus of thepresent invention has the capacity to undergo gene rearrangement andgene conversion and hypermutation in the non-human animal, therebyproducing a diversified repertoire of humanized Ig molecules. An Iglocus having the capacity to undergo gene rearrangement and geneconversion is also referred to as a “functional” Ig locus and theantibodies with a diversity generated by a functional Ig locus are alsoreferred to as “functional” antibodies or a “functional” repertoire ofantibody molecules.

In a further aspect, the invention provides nucleic acid moleculescomprising a human Ig gene segment, flanked by nucleotide sequenceswhich comprise at least bout 20 contiguous nucleotides from a spacersequence separating the coding regions in a non-primate animal Ig heavyor light chain gene, or from a consensus sequence of two or more of suchspacer sequences. The flanking sequences just as the spacersequence-derived sections within the flanking sequences can be identicalor different. The contiguous nucleotides derived from a spacer sequenceor from a consensus sequence of two or more spacer sequences can befused directly to the human Ig gene segment. Alternatively, there mightbe an intervening sequence between the human Ig gene segment and atleast one of the spacer-originating nucleotide sequences. Thus, forexample, a flanking sequence at the 5′ end of a human V gene segment mayinclude a promoter region, which is linked directly to the human V genesegment, and separates it from the spacer-sequence derived nucleotidestretch of at least 20 nucleotides.

In yet another aspect, the invention concerns a humanized Ig heavy chainlocus in which human heavy chain V, D and/or J gene segments and/or Cregion segments are present in the same configuration as in the originalnon-human animal immunoglobulin gene, and separated by sequencesincluding at least about 20 contiguous nucleotides from an intronsequence separating the coding regions in a non-primate animal Ig heavyor light chain gene. In another embodiment, the present inventionprovides a humanized light chain locus in which human light chain Cregion segments and/or J gene segments and/or V region segments areseparated by non-human animal (e.g. non-primate) intron sequences in thesame configuration as in the original non-human animal immunoglobulingene. In a particular embodiment, the spacer sequences are designedbased on non-coding, e.g. intron sequences of the non-human(non-primate) animal. In one embodiment, the spacers may retain theappropriate non-coding sequences from the non-human (non-primate)animal. Alternatively, in order to simplify the construct, a consensussequence, designed based upon the highly homologous non-coding (intron)sequences may be designed, and used as a uniform spacer sequence for thepreparation of multiple human heavy or light chain gene segments.

The invention specifically provides isolated nucleic acid sequences andvectors useful in the construction of humanized immunoglobulin loci.

In one embodiment, DNA fragments containing an Ig locus to be humanizedare isolated from animals which generate antibody diversity by geneconversion, e.g., rabbit and chicken. Such large DNA fragments can beisolated by screening a library of plasmids, cosmids, yeast artificialchromosomes (YACs) or bacterial artificial chromosomes (BACs), and thelike, prepared from the genomic DNA of the non-human, e.g. non-primateanimal. An entire animal C-region can be contained in one plasmid orcosmid clone which is subsequently subjected to humanization. YAC clonescan carry DNA fragments of up to 2 megabases, thus an entire animalheavy chain locus or a large portion thereof can be isolated in one YACclone, or reconstructed to be contained in one YAC clone. BAC clones arecapable of carrying DNA fragments of smaller sizes (about 150-450 kb).However, multiple BAC clones containing overlapping fragments of an Iglocus can be separately humanized and subsequently injected togetherinto an animal recipient cell, wherein the overlapping fragmentsrecombine in the recipient animal cell to generate a continuous Iglocus.

Human Ig gene segments can be integrated into the Ig locus on a vector(e.g., a BAC clone) by a variety of methods, including ligation of DNAfragments, or insertion of DNA fragments by homologous recombination.Integration of the human Ig gene segments is done in such a way that thehuman Ig gene segment is operably linked to the host animal sequence inthe transgene to produce a functional humanized Ig locus, i.e., an Iglocus capable of gene rearrangement and gene conversion andhypermutation which lead to the production of a diversified repertoireof humanized antibodies.

In one embodiment, human Ig gene segments can be integrated into the Iglocus by homologous recombination. Homologous recombination can beperformed in bacteria, yeast and other cells with a high frequency ofhomologous recombination events. For example, a yeast cell istransformed with a YAC containing an animal's Ig locus or a largeportion thereof. Subsequently, such yeast cell is further transformedwith a recombination vector as described hereinabove, which carries ahuman Ig gene segment linked to a 5′ flanking sequence and a 3′ flankingsequence. The 5′ and the 3′ flanking sequences in the recombinationvector are homologous to those flanking sequences of the animal Ig genesegment on the YAC. As a result of a homologous recombination, theanimal Ig gene segment on the YAC is replaced with the human Ig genesegment. Alternatively, a bacterial cell such as E. coli is transformedwith a BAC containing an animal's Ig locus or a large portion thereof.Such bacterial cell is further transformed with a recombination vectorwhich carries a human Ig gene segment linked to a 5′ flanking sequenceand a 3′ flanking sequence. The 5′ and the 3′ flanking sequences in therecombination vector mediate homologous recombination and exchangebetween the human Ig gene segment on the recombination vector and theanimal Ig gene segment on the BAC. Humanized YACs and BACs can bereadily isolated from the cells and used in making transgenic animals.

In a further aspect of the present invention, methods of makingtransgenic animals capable of producing humanized immunoglobulins areprovided.

According to the present invention, a transgenic animal capable ofmaking humanized immunoglobulins are made by introducing into arecipient cell or cells of an animal one or more of the transgenicvectors described herein above which carry a humanized Ig locus, andderiving an animal from the genetically modified recipient cell orcells.

The recipient cells may, for example, be from non-human animals whichgenerate antibody diversity by gene conversion and/or hypermutation,e.g., bird (such as chicken), rabbit, cows and the like. In suchanimals, the 3′proximal V gene segment is preferentially used for theproduction of immunoglobulins. Integration of a human V gene segmentinto the Ig locus on the transgene vector, either by replacing the3′proximal V gene segment of the animal or by being placed in closeproximity of the 3′proximal V gene segment, results in expression ofhuman V region polypeptide sequences in the majority of immunoglobulins.Alternatively, a rearranged human V(D)J segment may be inserted into theJ locus of the immunoglobulin locus on the transgene vector.

The transgenic vectors containing a humanized Ig locus is introducedinto the recipient cell or cells and then integrated into the genome ofthe recipient cell or cells by random integration or by targetedintegration.

For random integration, a transgenic vector containing a humanized Iglocus can be introduced into an animal recipient cell by standardtransgenic technology. For example, a transgenic vector can be directlyinjected into the pronucleus of a fertilized oocyte. A transgenic vectorcan also be introduced by co-incubation of sperm with the transgenicvector before fertilization of the oocyte. Transgenic animals can bedeveloped from fertilized oocytes. Another way to introduce a transgenicvector is by transfecting embryonic stem cells and subsequentlyinjecting the genetically modified embryonic stem cells into developingembryos. Alternatively, a transgenic vector (naked or in combinationwith facilitating reagents) can be directly injected into a developingembryo. Ultimately, chimeric transgenic animals are produced from theembryos which contain the humanized Ig transgene integrated in thegenome of at least some somatic cells of the transgenic animal.

In a particular embodiment, a transgene containing a humanized Ig locusis randomly integrated into the genome of recipient cells (such asfertilized oocyte or developing embryos) derived from animal strainswith an impaired expression of endogenous immunoglobulin genes. The useof such animal strains permits preferential expression of immunoglobulinmolecules from the humanized transgenic Ig locus. Examples for suchanimals include the Alicia and Basilea rabbit strains, as well asAgammaglobinemic chicken strain, as well as immunoglobulin knock-outmice. Alternatively, transgenic animals with humanized immunoglobulintransgenes or loci can be mated with animal strains with impairedexpression of endogenous immunoglobulins. Offspring homozygous for animpaired endogenous Ig locus and a humanized transgenic Ig locus can beobtained.

For targeted integration, a transgenic vector can be introduced intoappropriate animal recipient cells such as embryonic stem cells oralready differentiated somatic cells. Afterwards, cells in which thetransgene has integrated into the animal genome and has replaced thecorresponding endogenous Ig locus by homologous recombination can beselected by standard methods See for example, Kuroiwa et al, NatureGenetics 2004, Jun. 6. The selected cells may then be fused withenucleated nuclear transfer unit cells, e.g. oocytes or embryonic stemcells, cells which are totipotent and capable of forming a functionalneonate. Fusion is performed in accordance with conventional techniqueswhich are well established. Enucleation of oocytes and nuclear transfercan also be performed by microsurgery using injection pipettes. (See,for example, Wakayama et al., Nature (1998) 394:369). The resulting eggcells are then cultivated in an appropriate medium, and transferred intosynchronized recipients for generating transgenic animals.Alternatively, the selected genetically modified cells can be injectedinto developing embryos which are subsequently developed into chimericanimals.

Further, according to the present invention, a transgenic animal capableof producing humanized immunoglobulins can also be made by introducinginto a recipient cell or cells, one or more of the recombination vectorsdescribed herein above, which carry a human Ig gene segment, linked to5′ and 3′ flanking sequences that are homologous to the flankingsequences of the endogenous Ig gene segment, selecting cells in whichthe endogenous Ig gene segment is replaced by the human Ig gene segmentby homologous recombination, and deriving an animal from the selectedgenetically modified recipient cell or cells.

Similar to the target insertion of a transgenic vector, cellsappropriate for use as recipient cells in this approach includeembryonic stem cells or already differentiated somatic cells. Arecombination vector carrying a human Ig gene segment can be introducedinto such recipient cells by any feasible means, e.g., transfection.Afterwards, cells in which the human Ig gene segment has replaced thecorresponding endogenous Ig gene segment by homologous recombination,can be selected by standard methods. These genetically modified cellscan serve as nuclei donor cells in a nuclear transfer procedure forcloning a transgenic animal. Alternatively, the selected geneticallymodified embryonic stem cells can be injected into developing embryoswhich can be subsequently developed into chimeric animals.

Transgenic animals produced by any of the foregoing methods form anotherembodiment of the present invention. The transgenic animals have atleast one, i.e., one or more, humanized Ig loci in the genome, fromwhich a functional repertoire of humanized antibodies is produced.

In a specific embodiment, the present invention provides transgenicrabbits having one or more humanized Ig loci in the genome. Thetransgenic rabbits of the present invention are capable of rearrangingand gene converting the humanized Ig loci, and expressing a functionalrepertoire of humanized antibodies.

In another specific embodiment, the present invention providestransgenic chickens having one or more humanized Ig loci in the genome.The transgenic chickens of the present invention are capable ofrearranging and gene converting the humanized Ig loci, and expressing afunctional repertoire of humanized antibodies In another specificembodiment, the present invention provides transgenic mice with one ormore humanized V regions in the genome. The humanized V region comprisesat least two human V gene segments flanked by non-human spacersequences. The transgenic mice are capable of rearranging the human Velements and expressing a functional repertoire of antibodies.

Once a transgenic non-human animal capable of producing diversifiedhumanized immunoglobulin molecules is made, humanized immunoglobulinsand humanized antibody preparations against an antigen can be readilyobtained by immunizing the animal with the antigen. A variety ofantigens can be used to immunize a transgenic host animal. Such antigensinclude, without limitation, microorganisms, e.g. viruses andunicellular organisms (such as bacteria and fungi), alive, attenuated ordead, fragments of the microorganisms, or antigenic molecules isolatedfrom the microorganisms.

Exemplary bacterial antigens for use in immunizing an animal includepurified antigens from Staphylococcus aureus such as capsularpolysaccharides type 5 and 8, recombinant versions of virulence factorssuch as alpha-toxin, adhesin binding proteins, collagen bindingproteins, and fibronectin binding proteins. Exemplary bacterial antigensalso include an attenuated version of S. aureus, Pseudomonas aeruginosa,enterococcus, enterobacter, and Klebsiella pneumoniae, or culturesupernatant from these bacteria cells. Other bacterial antigens whichcan be used in immunization include purified lipopolysaccharide (LPS),capsular antigens, capsular polysaccharides and/or recombinant versionsof the outer membrane proteins, fibronectin binding proteins, endotoxin,and exotoxin from Pseudomonas aeruginosa, enterococcus, enterobacter,and Klebsiella pneumoniae.

Exemplary antigens for the generation of antibodies against fungiinclude attenuated version of fungi or outer membrane proteins thereof,which fungi include, but are not limited to, Candida albicans, Candidaparapsilosis, Candida tropicalis, and Cryptococcus neoformans.

Exemplary antigens for use in immunization in order to generateantibodies against viruses include the envelop proteins and attenuatedversions of viruses which include, but are not limited to respiratorysynctial virus (RSV) (particularly the F-Protein), Hepatitis C virus(HCV), Hepatits B virus (HBV), cytomegalovirus (CMV), EBV, and HSV.

Therapeutic antibodies can be generated for the treatment of cancer byimmunizing transgenic animals with isolated tumor cells or tumor celllines; tumor-associated antigens which include, but are not limited to,Her-2-neu antigen (antibodies against which are useful for the treatmentof breast cancer); CD19, CD20, CD22 and CD53 antigens (antibodiesagainst which are useful for the treatment of B cell lymphomas), (3)prostate specific membrane antigen (PMSA) (antibodies against which areuseful for the treatment of prostate cancer), and 17-1A molecule(antibodies against which are useful for the treatment of colon cancer).

The antigens can be administered to a transgenic host animal in anyconvenient manner, with or without an adjuvant, and can be administeredin accordance with a predetermined schedule.

After immunization, serum or milk from the immunized transgenic animalscan be fractionated for the purification of pharmaceutical gradepolyclonal antibodies specific for the antigen. In the case oftransgenic birds, antibodies can also be made by fractionating eggyolks. A concentrated, purified immunoglobulin fraction may be obtainedby chromatography (affinity, ionic exchange, gel filtration, etc.),selective precipitation with salts such as ammonium sulfate, organicsolvents such as ethanol, or polymers such as polyethyleneglycol.

For making a monoclonal antibody, spleen cells are isolated from theimmunized transgenic animal and used either in cell fusion withtransformed cell lines for the production of hybridomas, or cDNAsencoding antibodies are cloned by standard molecular biology techniquesand expressed in transfected cells. The procedures for making monoclonalantibodies are well established in the art. See, e.g., European PatentApplication 0 583 980 A1 (“Method For Generating Monoclonal AntibodiesFrom Rabbits”), U.S. Pat. No. 4,977,081 (“Stable Rabbit-Mouse HybridomasAnd Secretion Products Thereof”), WO 97/16537 (“Stable Chicken B-cellLine And Method of Use Thereof”), and EP 0 491 057 B1 (“Hybridoma WhichProduces Avian Specific Immunoglobulin G”), the disclosures of which areincorporated herein by reference. In vitro production of monoclonalantibodies from cloned cDNA molecules has been described byAndris-Widhopf et al., “Methods for the generation of chicken monoclonalantibody fragments by phage display”, J Immunol Methods 242:159 (2000),and by Burton, D. R., “Phage display”, Immunotechnology 1:87 (1995), thedisclosures of which are incorporated herein by reference.

Cells derived from the transgenic animals of the present invention, suchas B cells or cell lines established from a transgenic animal immunizedagainst an antigen, are also part of the present invention.

In a further aspect of the present invention, methods are provided fortreating a disease in a primate, in particular, a human subject, byadministering a purified humanized antibody composition, preferably, ahumanized polyclonal antibody composition, desirable for treating suchdisease.

In another aspect of the present invention, purified monoclonal orpolyclonal antibodies are admixed with an appropriate pharmaceuticalcarrier suitable for administration in primates especially humans, toprovide pharmaceutical compositions. Pharmaceutically acceptablecarriers which can be employed in the present pharmaceuticalcompositions can be any and all solvents, dispersion media, isotonicagents and the like. Except insofar as any conventional media, agent,diluent or carrier is detrimental to the recipient or to the therapeuticeffectiveness of the antibodies contained therein, its use in thepharmaceutical compositions of the present invention is appropriate. Thecarrier can be liquid, semi-solid, e.g. pastes, or solid carriers.Examples of carriers include oils, water, saline solutions, alcohol,sugar, gel, lipids, liposomes, resins, porous matrices, binders,fillers, coatings, preservatives and the like, or combinations thereof.

The humanized polyclonal antibody compositions used for administrationare generally characterized by containing a polyclonal antibodypopulation, having immunoglobulin concentrations from 0.1 to 100 mg/ml,more usually from 1 to 10 mg/ml. The antibody composition may containimmunoglobulins of various isotypes. Alternatively, the antibodycomposition may contain antibodies of only one isotype, or a number ofselected isotypes.

In most instances the antibody composition consists of unmodifiedimmunoglobulins, i.e., humanized antibodies prepared from the animalwithout additional modification, e.g., by chemicals or enzymes.Alternatively, the immunoglobulin fraction may be subject to treatmentsuch as enzymatic digestion (e.g. with pepsin, papain, plasmin,glycosidases, nucleases, etc.), heating, etc, and/or furtherfractionated.

The antibody compositions generally are administered into the vascularsystem, conveniently intravenously by injection or infusion via acatheter implanted into an appropriate vein. The antibody composition isadministered at an appropriate rate, generally ranging from about 10minutes to about 24 hours, more commonly from about 30 minutes to about6 hours, in accordance with the rate at which the liquid can be acceptedby the patient. Administration of the effective dosage may occur in asingle infusion or in a series of infusions. Repeated infusions may beadministered once a day, once a week once a month, or once every threemonths, depending on the half-life of the antibody preparation and theclinical indication. For applications on epithelial surfaces theantibody compositions are applied to the surface in need of treatment inan amount sufficient to provide the intended end result, and can berepeated as needed. In addition, antibodies can, for example, beadministered as an intramuscular bolus injection, which may, but doesnot have to, be followed by continuous administration, e.g. by infusion.

The antibody compositions can be used to bind and neutralize antigenicentities in human body tissues that cause disease or that elicitundesired or abnormal immune responses. An “antigenic entity” is hereindefined to encompass any soluble or cell-surface bound moleculesincluding proteins, as well as cells or infectious disease-causingorganisms or agents that are at least capable of binding to an antibodyand preferably are also capable of stimulating an immune response.

Administration of an antibody composition against an infectious agent asa monotherapy or in combination with chemotherapy results in eliminationof infectious particles. A single administration of antibodies decreasesthe number of infectious particles generally 10 to 100 fold, morecommonly more than 1000-fold. Similarly, antibody therapy in patientswith a malignant disease employed as a monotherapy or in combinationwith chemotherapy reduces the number of malignant cells generally 10 to100 fold, or more than 1000-fold. Therapy may be repeated over anextended amount of time to assure the complete elimination of infectiousparticles, malignant cells, etc. In some instances, therapy withantibody preparations will be continued for extended periods of time inthe absence of detectable amounts of infectious particles or undesirablecells. Similarly, the use of antibody therapy for the modulation ofimmune responses may consist of single or multiple administrations oftherapeutic antibodies. Therapy may be continued for extended periods oftime in the absence of any disease symptoms.

The subject treatment may be employed in conjunction with chemotherapyat dosages sufficient to inhibit infectious disease or malignancies. Inautoimmune disease patients or transplant recipients, antibody therapymay be employed in conjunction with immunosuppressive therapy at dosagessufficient to inhibit immune reactions. The invention is furtherillustrated, but by no means limited, by the following examples.

EXAMPLE 1 Isolation and Sequencing of BAC Clones Containing RabbitImmunoglobulin Loci

High molecular weight DNA was isolated from a2b5 male rabbits. Therabbits were euthanized, spleen and kidneys were removed and rinsed inice-cold PBS. Fat and connecting tissues were removed and processedseparately. The organs were cut into pieces and homogenized in apre-cooled Dounce homogenizer. The supernatant was transferred intocooled 50 ml falcon tubes, mixed with cold PBS and large tissue debriswas allowed to sink to the bottom for 2 minutes. Cells in thesupernatant were pelleted at 200 g for 10 min at 4° C., washed once withPBS, resuspended in 1 ml PBS and counted. Sets of 5×10⁶, 5×10⁷ and 5×10⁸cells were embedded in agarose plugs using the CHEF Mammalian GenomicDNA Plug Kit (BIORAD) To optimize conditions for partial digestion withHindIII, plugs were cut into 5 equal pieces and digested with 1 to 10units of HindIII for various times and temperatures. Best results wereobtained with 2 units HindIII at 4° C. for 3 hrs or 37° C. for 25 min.Digested DNA was double size fractioned on a Pulse Field GelElectrophoresis (PFGE) apparatus using the following parameters: 6 hrbackwards, 15 s switch times; 6 hr forwards, 15 s switch times; 20 hrforwards, 90 s switch times; 200V 14° C. The area of the gel with thedesired size of partial digested DNA was cut and DNA was isolated usinggelase. 11 ng of insert was ligated with 1 ng of HindIII digestedpBELOBAC 11 and electroporated into DH10B cells. 1% of the resultingcolonies was sized using NotI and revealed an average insert size of 124kb. 1×10⁵ clones were spotted on Nylon filters and screened byhybridization with specific probes.

Probes for screening were amplified by PCR using genomic DNA fromrabbits, cloned into pBlueScript, and verified by sequencing. Primerpairs (SEQ ID NO: 193-208, Table 2) were designed according to publishedsequences. Several BACs representing rabbit heavy and light chainimmunoglobulin loci were isolated and mapped (FIGS. 1 and 5). BACs219D23 219D23 (GenBank Acc. No. AY386695), 225P18 (GenBank Acc. No.AY386697), 27N5 (GenBank Acc. No. AY386696), 38A2 (GenBankAcc. No.AY386694), 179L1 (GenBank Acc. No. AY495827), 215M22 (GenBank Acc. No.AY495826), 19 (GenBankAcc. No. AY495828) and Fosmid Fos15B (GenBank Acc.No. AY3866968) were sequenced. Shotgun libraries for sequencing wereconstructed in pCR-Blunt with an insert size of 1.5-2 kb. For sequenceanalysis the STADEN package (Roger Staden, Cambridge, UK) was used. Thesoftware modules pregap and gap4 were used for assembly and gap closure.For the quality clipping of sequences PHRED (Washington University) andthe STADEN package was coupled.

EXAMPLE 2 Construction of a Humanized Rabbit Immunoglobulin Heavy ChainLocus

BAC and fosmid clones containing rabbit immunoglobulin heavy chain locussequences were isolated from genomic DNA libraries using probes specificfor the constant, variable, and joining gene segments or the 3′ enhancerregion. Isolated BACs (FIG. 1) 27N5 (GenBank Acc. No. AY386696), 219D23(GenBankAcc. No. AY386695), 225P18 (GenBank Acc. No. AY386697), 38A2(GenBank Acc. No. AY386694) and fosmid Fos15B (GenBank Acc. No.AY3866968) were sequenced (Ros et al., Gene 330, 49-59).

Selected immunoglobulin coding sequences were exchanged withcorresponding human counterparts by homologous recombination in E. Coliby ET cloning (E-Chiang Lee et al., Genomics 73, 56-65 (2001); DaiguanYu et al., PNAS 97, 5978-5983 (2000); Muyrers et al., Nucleic AcidsResearch 27, 1555-1557 (1999); Zhang et al., Nature Biotechnology 18,1314-1317 (2000)).

Alternatively, DNA fragments were recombined by ligation in vitro andsubsequent transformation of E. coli. BACs and/or Fos15B or partsthereof were combined by in vitro ligation and transformation, ETcloning, or by Cre recombinase mediated integration.

For ET cloning, vectors containing target sequence were transformed intoa streptomycin resistant E. coli strain containing the inducible lambdaphage recombination enzymes Redα, Redβ and γ. These recombinationproteins were expressed either from a co-transfected plasmid (DH10B E.coli cells with plasmid pSC101) or from a genomic integrated lambdaprophage (DY380 E. coli strain). The ET cloning procedure encompassedtwo homologous recombination steps.

In a first step the target locus was replaced by a selection-counterselection cassette (e.g. neo-rpsL which confers resistance to neomycin(neo) and sensitivity to streptomycin (rpsL)). After isolation ofneo-resistant colonies, insertion of the selection cassette byhomologous recombination was confirmed by restriction enzyme analysisand partial sequencing.

In a second step, the rpsL-neo selection cassette was exchanged with anew sequence. Streptomycin resistant clones were analyzed by restrictionanalysis and sequencing. Fragments used for the ET cloning procedure hadflanking sequences of 20 to 50 bp length, which were identical to targetsequences. Sequences used for ligation had appropriate restrictionenzyme sites at their 3′ and 5′ ends. These sites were either naturallyoccurring sites or they were introduced by PCR using primers containingappropriate sites.

Alternatively, sequences were generated synthetically.

A humanized heavy chain was constructed by replacement of rabbit J_(H),Cμ in BAC 219D23 and Cγ in BAC 27N5 with their corresponding humancounterparts by ET cloning. Human sequences used for the ET cloningprocedures were amplified by PCR from human genomic DNA.

Human Cμ, Cγ and J_(H) gene segments was amplified using primers (SEQ IDNos: 209-214, Table 2) with 50 bp homologies to rabbit target sequences.

After ligation of BAC clone 225P18 with clone 219D23 and BAC 27N5 withFosmid 15B, the ligated constructs were transformation into E. coli andconnected by Cre recombinase mediated insertion. This resulted in afunctional locus consisting of 18 rabbit variable genes, rabbit Dregion, human J region, human Cμ, human Cγ, rabbit Cϵ, rabbit Cα4 andthe 3′enhancer element.

For the generation of transgenic animals the humanized BAC clones werecoinjected either separately as three overlapping BACs (225P18 and219D23 and BAC 27N5) or two overlapping combined BACs (225P18-219D23 andBAC 27N5-Fosmid 15B) or as one BAC (225P18-219D23-27N5-Fosmid 15B).Founder animals with transgenes were identified by PCR.

EXAMPLE 3 Construction of a Humanized Immunoglobulin Heavy Chain LocusUsing Synthetic Fragments

Four fragments denoted Unit1, Unit2, Unit3, and Unit 4 (FIG. 13, SEQ IDNos: 189-192) with human V sequences and rabbit spacers were chemicallysynthesized. Each fragment was flanked 5′ by an AscI restrictionendonuclease recognition sequence, 3′ by a lox71 Cre recombinaserecognition sequence followed by Fse I and MluI restriction enzymerecognition sequences. Unit 1 consisted of human V_(H)3-49, V_(H)3-11,V_(H)3-7 and V_(H)3-15 variable genes separated by rabbit spacersI29-30, I3-4, I2-3 and the 3′ half of I1-2 (I1-2B). Unit 2 consisted ofhuman V_(H)3-48, V_(H)3-43 and V_(H)3-64 separated by rabbit spacersI1-2A (5′ half of I1-2), I7-8, I6-7 and the 3′ half of I4-5 (I4-5B).Unit 3 consisted of human V_(H)3-74, V_(H)3-30, and V_(H)3-9 separatedby the rabbit spacer sequences I4-5B, I26-27, I11-12 and I17-18.

Unit 3 had in addition to the afore mentioned upstream flanks an Flprecombinase recognition target (FRT) sequence, followed by a Sglf Irestriction endonuclease recognition sequence preceding the alreadymentioned Asc I site.

Unit 4 had the human V_(H)3-23 gene 5′ flanked by the rabbit spacerI1-2, a lox66 Cre recombinase target sequence and an AscI endonucleaserecognition sequence, and 3′ flanked by IV-C (5′ half) rabbit spacersequence followed by a MluI endonuclease recognition sequence.

A gentamycin selection cassette was PCR-amplified, using primers SEQ IDNOs 215 and 216 (Table 2) containing AscI and FseI sites and ligatedinto a pGEM vector with a modified cloning site including AscI, FseI,and MluI endonuclease recognition sites (pGEM.Genta modified by PCRusing SEQ ID NOs 217 and 218, Table 2).

Units 1, 2 and 3 were cloned into pGEM.Genta (Promega) vectors.

Unit 4 was sub-cloned into a customized pBELOBAC11 (NEB) vectorlinearized with Hind III, and PCR-amplified. The forward primer (SEQ IDNO: 219, Table 2) had restriction sites for HindIII, PacI and AatII, andthe reverse primer (SEQ ID NO: 220) sites for Bam HI, MluI and AscI. Theprimers were designed in such a way that the pBELOBAC11 Chloramphenicolselection cassette was deleted. Furthermore, a Neomycin selectioncassette was PCR-amplified with primers SEQ ID NOs 221 and 222 (Table 2)carrying Bam HI and Hind III restriction sites, and ligated to themodified pBELOBAC11 vector (pBB11.1).

Units 1-4 were assembled by cre-mediated recombination as described(Mejia et al, Genomics 70(2) 165-70 (2000)). First Unit 1 was clonedinto the customized pgem.Genta vector, digested with Fse I andsubsequently recircularized by ligation. This vectorless construct wastransformed into E. coli containing pBB11.1.Unit4 and p706-Cre plasmid.Following recombination of Unit 1 with PBB11.1 unit 4, positive clones(Unit4/1) were selected on kanamycin and gentamycin containing media.Clones were characterized by restriction analyses using various enzymes.

For recombination of Unit 2, the Unit4/1 insert was excised by doubledigestion with AscI and PacI, and cloned into pBELOBAC11 with a modifiedlinker (pBB11.2: modified by PCR using primers SEQ ID NOs 223 and 224,Table 2).

pBELOBAC11 was linearized with HindIII and PCR-amplified with a forwardprimer encoding PacI and AatII endonuclease recognition sites and areverse primer encoding MluI and NotI endonuclease recognition sites anda lox66 Cre recombinase target site. For ligation with Unit1/4 thepBB11.2 vector was opened with MluI and PacI. pGEM.Genta.Unit2 wasconverted into a circular vectorless construct as described forpGEM.Genta.Unit1 and connected with pBB11.2.Unit4/1 by in vivo Cremediated recombination. Subsequently, the resulting constructpBB11.2.Unit4/1/2 is prepared for Cre mediated recombination with Unit 3by replacing the wild type loxp site with a lox66 target site byET-cloning (Muyrers et al., Nucleic Acids Research 27, 1555-1557 (1999);Muyrers et at Trends Biochem. Sci. 26(5):325-31 (2001)). Achloramphenicol selection cassette was amplified by PCR with primers(SEQ ID NOs 225 and 226, Table 2) containing 50 bp sequences homologousto the BAC target sequence. The reverse primer included a lox66 site.The gel-purified PCR product was transformed into cells carrying thetarget BAC as well as the pSC101 plasmid, required for homologousrecombination. Positive clones were selected with chloramphenicol andconfirmed by restriction analysis and sequencing. pGEM.Genta.Unit 3 wasprepared for in vivo recombination as described above for Unit1 and 2and transformed into cells carrying the receptor BAC, as well as thep706-Cre plasmid. Positive clones pBB11.2.Unit4/1/2/3 were selected withgentamycin and confirmed by restriction analysis. pBB11.2.Unit4/1/2/3was further modified by ET-cloning to generate a lox 71 target site.Subsequently, pBB11.2.Unit4/1/2/3 was connected to fragments from BACs219D23, 27N5 and Fos15B.

EXAMPLE 4 Construction of a Humanized Immunoglobulin Heavy Chain LocusUsing PCR Amplified Fragments

Human V_(H) elements were amplified using genomic DNA (ClonTech) andprimers SEQ ID NOs 227-248 (Table 2). PCR products were analyzed bygel-electrophoresis and gel purified using the GENECLEAN kit (Q-Biogen).Subsequently, amplification products were sub-cloned into Zero-BluntTOPO™ (Invitrogen), according to the manufacturer's instructions. Thesequences of all amplified V elements were confirmed. For theconstruction of the humanized V region, V elements were amplified usingplasmid DNA as template and primers SEQ ID NOs 249-270 (Table 2).Forward primers contained an AscI site, followed by a rabbit splicesite. Reverse primers contained a rabbit recombination signal sequence(RSS) and a MluI restriction site. PCR products were gel purified usingthe GENECLEAN kit.

Human V_(H)s, V3-33, V3-74, V3-49, V3-21, V3-48, V3-73, V3-7, and V3-Dcould not be isolated by PCR and were synthesized chemically (BlueHeron,Bothel, Wash.). Restriction sites and rabbit regulatory sequences wereadded during synthesis.

Rabbit spacer sequences were amplified using BACs 38A2 and 225P18 astemplates and primers SEQ ID NOs 271-288 (Table 2). BAC 225P18 wasdouble digested with NheI and a 41 kb fragment was gel purified. Thisfragment served as a template for the amplification of spacers V1-2,V2-3, V3-4, V4-5, and V5-6.

BAC 225P18 was digested with BstBI and the template for spacers V6-7 andV7-8 was gel-purified. A double digestion of BAC 38A2 with PacI andRsrlI allowed gel purification of the template for spacers V21-22, andV22-23.

Amplified spacer sequences were gel-purified, and subcloned intoXL-PCR-TOPO™ (Invitrogen) according to the manufacturer's instructions.

V_(H) elements and rabbit spacer sequences were sub-cloned into modifiedpGEM (Promega) and pBS (Strategene) vectors. The pGEM vector was cutwith NotI and Hind III and ligated with chemically synthesizedoligonucleotide sequences containing FseI, AscI and MluI sites (Oligo1:SEQ ID NO: 289; Oligo2: SEQ ID NO: 290; Table 2). Vector pBS was cutwith SacI and KpnI and ligated with a chemically synthesizedoligonucleotide sequence containing the restriction sites FseI, AscI andMluI (Oligo1: (SEQ ID NO: 291; Oligo2: SEQ ID NO: 292, Table 2).

Gentamycin and neomycin selection cassettes were amplified using primers(SEQ ID NOs: 293-296, Table 2) with Fse I or AscI sites and ligated intothe modified pGEM and pBS-vectors.

The final construct is built in a modified pBeloBAC II vector. ThepBeloBAC II vector was opened with BamHI and HindIII and the cloningsites were modified to contain FseI, AscI, MluI sites using a chemicallysynthesized oligonucleotide sequence (Oligo1: SEQ ID NO: 297; Oligo2:SEQ ID NO: 298, Table 2).

BAC 219D23 was modified by introduction of restriction sites usingET-cloning (Muyrers et al., Nucleic Acids Research 27, 1555-1557 (1999);Muyers et at Trends Biochem. Sci. 26(5):325-31 (2001)). The Neomycinselection cassette was amplified with primers SEQ ID NO: 299 and SEQ IDNO: 300 (Table 2). The forward primer contained an FseI site, thereverse primer an AscI site.

The purified PCR product was transformed into E. coli cells carrying BAC219D23 and plasmid pSC101 necessary for homologous recombination. Afterhomologous recombination of the cassette and the target sites in theBAC, introduced restriction sites were confirmed by restrictionanalysis. Subsequently, the modified BAC 219D23 was digested with FseIand MluI and the resulting 17 kb fragment (containing the FseI-Neo-AscIcassette) was separated by PFGE and purified by electro-elution. Thispurified fragment was ligated with the modified pBeloBAC II vectoropened with FseI and MluI.

A purified DNA fragment encoding a human V_(H) element is ligated withthe modified pGEM.neo vector opened with AscI and MluI. Similarly aspacer sequence is sub-cloned into the modified pGEM.genta vector.Subsequently, the pGEM.genta vector carrying the spacer sequence is cutwith FseI and MluI and the insert is ligated with pGEM.neo.V_(H) vectoropened with FseI and AscI. This step is repeated several times to builda fragment consisting of several spacer and V_(H) segments. Suchfragments are excised with FseI and MluI and ligated with the modifiedpBeloBAC II vector linearized with FseI and AscI. These processes arerepeated to build a large immunoglobulin V region (FIG. 6). Thehumanized heavy chain locus is used for the generation of transgenicanimals.

EXAMPLE 5 Construction of a Humanized Rabbit Light Chain LocusContaining Humanized Ck and Humanized Rearranged VJ

Screening of a rabbit genomic BAC libraries resulted in theidentification of three BACs (215M22, 179L1 and 196O2; Gene BankAccession Nos: AY495826, AY495827, and AY495828, respectively)containing rabbit light chain K1 gene segments. Rabbit Cκ□ was exchangedwith human Cκ□ allotype Km3 by ET cloning as described (E-Chiang Lee etal., Genomics 73, 56-65 (2001); Daiguan Yu et al., PNAS 97, 5978-5983(2000); Muyrers et al., Nucleic Acids Research 27, 1555-1557 (1999);Zhang et al., Nature Biotechnology 18, 1314-1317 (2000)). Human Cκ(allotype Km3) was amplified by PCR with primers (SEQ ID Nos 301 and302, Table 2) containing 50 bp sequences homologous to target sequences.Homology arms were designed based on the published sequence of rabbitgermline kappa (b5; GenBank Accession No. K01363) and matched theintron-exon boundary of Cκ. The exchange of rabbit Cκagainst the humanCκ in BAC 179L1 was verified by sequencing.

BAC 179L1-huCk was modified by two ET cloning. A neomycin selectioncassette was amplified with primers (SEQ ID NOs 303 and 304, Table 2)containing 50 bp sequences homologous to BAC 179L1. The forward primeradditionally had an i-CeuI meganuclease site. The PCR product was usedfor ET cloning. Positive clones were selected with neomycin and checkedfor correctness by restriction enzyme digests and sequencing. A zeocinselection cassette was amplified with primers (SEQ ID NOs 305 and 306,Table 2) containing 50 bp sequences homologous to BAC 179L1. The forwardprimer additionally had an i-SceI meganuclease site. The PCR product wasused for ET cloning. Positive clones were selected with zeozin andchecked for correctness by restriction enzyme digests and sequencing.

BAC 215M22 was modified by one ET cloning. A gentamycin resistance genewas amplified with primers (SEQ ID NOs 307 and 308, Table 2) containing50 bp sequences homologous to BAC215M22. The forward primer additionallyhad an i-CeuI Meganuclease site and the reverse primer an i-SceImeganuclease site. The PCR product was used for ET cloning. Resultingclones were selected with gentamycin and analyzed by restriction enzymedigests and sequencing.

Modified BAC179L1 and 225M22 were cut with i-CeuI and i-SceI. Fragmentsof 98 kb and 132 kb were purified and ligated. Resulting clones wereselected with kanamycin and chloramphenicol and checked for correctnessby restriction enzyme digests, PCR of the regions containing i-SceI andi-CeuI restriction sites, and sequencing. The resulting BAC was termed179-215-huCk.

Rabbit Jκ1 and Jκ2 of BAC 179-215-huCk were replaced by ET cloning witha synthetic human rearranged VκJκ gene. A DNA fragment with rabbitpromoter, rabbit leader, rabbit intron and human VκJκ gene wassynthesized chemically. The codon usage of the synthetic human VJ wasoptimised to achieve highest DNA sequence homology to rabbit V kappagenes.

The synthetic human VJ was PCR amplified with a forward primer (SEQ IDNO 309, Table 2) containing 50 bp sequences homologous to BAC 179L1 anda reverse primer (SEQ ID NO 310, Table 2) containing a sequencehomologous to the gentamycin resistance gene and a FRT site. Agentamycin resistance gene was amplified with a forward primer (SEQ IDNO 311, Table 2) containing a FRT site and a reverse primer (SEQ ID NO312, Table 2) with 50 bp homology to BAC 179L1 and a FRT site. The humansynthetic human VJ and the gentamycin resistance gene were combined byoverlap extension PCR using the forward primer for the synthetic humanVJ gene and the reverse primer for the gentamycin resistance gene. Theresulting fragment was used for ET cloning. Positive clones wereselected with gentamycin and checked for correctness by restrictionenzyme digests and sequencing.

The gentamycin resistance gene was removed by site specificrecombination via expression of Flp recombinase. After recombination oneFRT was left. The FRT site was deleted by ET cloning. A 232 bp fragmentfrom the synthetic human VJ was amplified by PCR (using primers SEQ IDNOs 313 and 314, Table 2) and used for ET cloning. Resulting colonieswere screened by PCR (using primers SEQ ID NOs 315 and 316, Table 2) forloss of the FRT site and confirmed by sequencing.

The neomycin resistance gene of BAC179-215-huCk was replaced by ETcloning. A gentamycin resistance (pRepGenta; Genebridges) gene wasamplified by PCR with primers (SEQ ID NOs 317 and 318, Table 2)containing 50 bp sequences homologous to BAC 179-215-huCk. The forwardprimer additionally had a loxP site, an attB site and a PvuI restrictionsite. Resulting clones were selected with gentamycin and checked forcorrectness by restriction enzyme digests and sequencing.

The resulting BAC (rLC3-B; FIG. 8) was used for the generation oftransgenic animals.

EXAMPLE 6 Construction of a Humanized Rabbit Light Chain LocusContaining Multipe Human Vk Elements, Chicken Spacer Elements and aRearranged Human VJ

Screening of a rabbit genomic BAC libraries resulted in theidentification of three BACs (215M22, 179L1 and 196O2; Gene BankAccession Nos: AY495826, AY495827, and AY495828, respectively)containing rabbit light chain K1 gene segments. Rabbit Cκ□0 wasexchanged with human Cκ□ allotype Km3 by ET cloning as described(E-Chiang Lee et al., Genomics 73, 56-65 (2001); Daiguan Yu et al., PNAS97, 5978-5983 (2000); Muyrers et al., Nucleic Acids Research 27,1555-1557 (1999); Zhang et al., Nature Biotechnology 18, 1314-1317(2000)). Human Cκ allotype Km3) was amplified by PCR with primers (SEQID Nos 301 and 302, Table 2) containing 50 bp sequences homologous totarget sequences. Homology arms were designed based on the publishedsequence of rabbit germline kappa (b5; GenBank Accession No. K01363) andmatched the intron-exon boundary of Cκ. The exchange of rabbit Cκagainst the human Cκ in BAC 179L1 was verified by sequencing.

Two DNA fragments, Unit1 (12,235 bp, FIG. 12a, SEQ ID NO 187),containing 17 human V pseudogenes and 18 chicken spacer sequences andUnit 2 (13,283 bp, FIG. 12b, SEQ ID NO 188) containing one functionalrearranged human kappa VJ gene with leader, 11 human V pseudogenes, 12chicken spacer sequences and intron 1 and parts of intron 2 weresynthesized chemically and cloned into vector pBR322.

Units 1 and 2 were digested with the restriction enzyme NgoMIV and AsiSIor NgoMIV and AscI respectively and ligated into pBELOBAC11 with amodified linker by three fragment ligation. The modified linkercontained a BsiWI restriction site, a FRT5-site, a rpsL-Neo-cassette, aAscI site and a AsiSI-site. The linker fragment was amplified with Highfidelity polymerase (Roche), primers CE_1_001_012904 (SEQ ID NO 319,Table 2) and CE_1_on005_013004 (SEQ ID NO 320, Table 2) and plasmidpRpsL-Neo (Genebridges) as template. Subsequently, the amplified productwas ligated into BamHI and HindIII sites of pBELOBAC11. For ligationwith Unit 1 and 2 the modified pBELOBAC11 was opened with AsiSI andAscI. Positive clones (pBELOBAC11 Unit1/2) were checked by restrictionenzyme digests.

BAC 179L1 (GENBANK Acc. No. AY495827) was modified by insertion of twomodified selection cassettes by ET cloning. Cassette 1 was a gentamycinresistance gene amplified with primers (SEQ ID Nos 321 and 322, Table 2)containing 50 bp sequences homologous to BAC 179L1 and an AscI site inthe reverse primer. Cassette 2 was a rpsl-Neo selection cassetteamplified with primers (SEQ ID Nos 323 and 324, Table 2) containing 50bp sequences homologous to BAC 179L1 and an attB site, a FRT5 site and aBsiWI site in the forward primer.

The purified PCR products were transformed into E. coli cells carryingthe BAC and plasmid pSC101 necessary for homologous recombination. Afterhomologous recombination successful modification of the BAC wasconfirmed by restriction digest analyses, Southern Blot and sequencing.

Modified BAC 179L1 was cut with the restriction enzymes AscI and BsiWI.The fragment containing the human Cκ was purified and ligated withpBELOBAC11 Unit1/2 opened with the same restriction enzymes. Positiveclones were checked by restriction enzyme digests. The final construct(FIG. 12c) is used for the generation of transgenic animals

EXAMPLE 7 Construction of a Humanized Rabbit Light Chain LocusContaining Multiple Human Vκ Elements, Chicken Spacers and anUnrearranged Human J Kappa Locus

The construct described in example 6 was modified by ET cloning asfollows: an. The rearranged functional VJ sequence was exchanged with afunctional V1 flanked by a functional recombination signal sequence(RSS). The RSS was PCR amplified from BAC179L1 with a forward primer(SEQ ID NO 325, Table 2) containing a 50 bp sequence homologous to V1 ofpBELOBAC11 Unit1/2 and a reverse primer (SEQ ID NO 326, Table 2)containing an AscI restriction enzyme site and homology to thegentamycin resistance gene. A gentamycin resistance gene was amplifiedwith a forward primer (SEQ ID NO 327, Table 2) containing a sequencehomologous to the reverse primer used for RSS amplification and areverse primer (SEQ ID NO 328, Table 2) containing a 50 bp sequencehomologous to pBELOBAC11 Unit1/2 and a BsiWI restriction enzyme site.

The RSS and the gentamycin resistance gene were combined by overlapextension PCR using the forward primer for RSS amplification and thereverse primer for Gentamycin resistance gene amplification. Theresulting fragment was used to modify pBELOBAC11 Unit1/2 by ET cloning.Positive clones were selected with gentamycin and analyzed byrestriction enzyme digests and sequencing.

BAC 179L1 with human Cκ was further modified by ET cloning. A kanamycinselection cassette was amplified with a primers (SEQ ID NO 329 and 330,Table 2) containing 50 bp sequences homologous to BAC 179L1. The reverseprimer contained also an AscI restriction enzyme site and a FRT site.The PCR product was used for ET cloning. An ampicillin selectioncassette was amplified with primers (SEQ ID Nos 331 and 332, Table 2)containing 50 bp sequences homologous to BAC 179L1. The forward primercontained also an attB site, an AsiSI restriction enzyme site and a FRT5site The reverse primer contained a BsiWI restriction enzyme site and aFRT site. The PCR product was used for ET cloning. The human J regionwas amplified from human genomic DNA with primers (SEQ ID Nos 333 and334, Table 2) containing 50 bp sequences homologous to BAC 179L1. ThePCR product was used for ET cloning. The resulting clones were analyzedby restriction enzyme digest and sequencing.

A positive clone was cut with AscI and BsiWI. The resulting fragment waspurified and ligated into the modified pBELOBAC11 Unit1/2 cut with thesame enzymes. Positive clones were selected with ampicillin and analyzedby restriction enzyme digests and sequencing. Correct clones are used togenerate transgenic animals.

EXAMPLE 8 Construction of a Humanized Rabbit Light Chain LocusContaining Multiple Human Vk Elements

Screening of a rabbit genomic BAC libraries resulted in theidentification of three BACs (215M22, 179L1 and 196O2; Gene BankAccession Nos: AY495826, AY495827, and AY495828, respectively)containing rabbit light chain K1 gene segments. Rabbit Cκ1 was exchangedwith human Cκ□ allotype Km3 by ET cloning as described (E-Chiang Lee etal., Genomics 73, 56-65 (2001); Daiguan Yu et al., PNAS 97, 5978-5983(2000); Muyrers et al., Nucleic Acids Research 27, 1555-1557 (1999);Zhang et al., Nature Biotechnology 18, 1314-1317 (2000)). Human Cκallotype Km3) was amplified by PCR with primers (SEQ ID NOs 301 and 302,Table 2) containing 50 bp sequences homologous to target sequences.Homology arms were designed based on the published sequence of rabbitgermline kappa (b5; GenBank Accession No. K01363) and matched theintron-exon boundary of Cκ. The exchange of rabbit Cκ against the humanCκ in BAC 179L1 was verified by sequencing.

Human Vκ elements of the Vκ1 family (O2, L8, L4, A30, L11, L1, L5, L15,O8, L19, L12, A20, O4, L14, L23, L9, A4, L24, O6, L22, A9, A25, A15, O9)were amplified by PCR using primers (SEQ ID NOs 335-382, Table 2) andhuman genomic DNA as a template.

Amplification products were analysed by gel-electrophoresis, gelpurified using GENECLEAN (Q-Biogen), subcloned into the Zero-Blunt TOPO™vectors (Invitrogen) and sequenced. A rearranged human Vκ (O2) Jκ (J4)element was produced by PCR amplification, subcloned and sequenced. Tocombine human Vκ elements with rabbit spacers, human Vκ elements wereamplified by PCR with primers (SEQ ID Nos 383-430, Table 2) usingplasmid DNA as a template. Primers contained AscI or MluI sites.

Rabbit spacer sequences are amplified by PCR using primers SEQ ID NOs431-450 (Table 2). BACs 179L1 and 215M22 are digested with SpeI, NheI,AclI, SfoI, MluI, and SalI/XhoI. Fragments are gel purified and used asamplification templates.

The spacer sequence located at the 5-end is amplified by an upstreamoligonucleotide containing a FRT and an attB site. PCR products are gelpurified using the GENECLEAN kit and subcloned into XL-PCR-TOPO™(Invitrogen) according to the manufacturer's instructions.

Human Vk elements and rabbit spacer sequences were cloned into pGem(Promega) modified as described in Example 4.

Human V kappa and the modified pGEM.genta vector are digested with AscIand MluI and ligated. Similarly, rabbit spacer sequences are cloned intopGEM.neo. Subsequently, pGem.neo. Vκ is cut with FseI and AscI andligated with a purified insert of pGem.genta.spacer excised with FseIand MluI. Ligation of AscI and MluI complementary ends destroys therestriction enzyme site and allows repeated use of AscI and MluI for theconstruction of a Vκ locus comprising several Vκ and spacer elements.The final construct, consisting of fragments of a humanized BAC 179L1and 215M22 and a humanized Vκ region is built in pBeloBAC. BAC 179L1 and215M22 were modified and combined. Subsequently, BAC 179L1-215M22-huCkwas further modified by ET cloning. Two cassettes containing restrictionenzyme site, selection markers, and additional functional sites wereinserted into the vector by ET-cloning as shown in FIG. 7. Primers (SEQID NOs 451-454) used for the amplification of the cassettes are listedin Table 2.

To built the final construct, units consisting of human V elements,rabbit spacer elements and a resistance marker are excised out of pGEMwith FseI and MluI and ligated with BAC 179L1-215M22 digested with FseIand AscI. Subsequently, the resistance marker is replaced with a newinsert consisting of human V elements, rabbit spacer elements andanother resistance marker. After several repeats the final constructwill consist of many Vk segments (L8, L4, A30, L11, L1, L5, L15, O8,L19, L12, A20, O4, L14, L23, L9, A4, L24, O6, L22, A9, A25, A15, O9)separated by rabbit spacer sequences. The humanized light chain locus isused for the generation of transgenic animals.

EXAMPLE 9 Construction of a Humanized Heavy Chain Locus with ChickenHeavy Chain Locus Spacer Sequences

A synthetic humanized heavy chain locus containing a rabbit D region, ahuman J region, human Cμ, human Cγ, rabbit Cα4, the rabbit 3′α enhancerand human VH elements (including promotor and nonamer/heptamersequences) separated by chicken spacer sequences is constructed.

Modified rabbit BAC 27N5 (see “Example 2) was further modified by ETcloning. The construct contained a humanized Cμ and Cγ and two uniquerestriction sites, BsiWI and AsiSI downstream of the α-4 membrane exon.DNA is amplified with oligonucleotides SEQ ID Nos 455 and 456 (Table 2).

Fosmid Fos15B is digested with NheI and the resulting 13 kb fragmentcontaining the 3′α enhancer is subcloned into a cloning vector in such away that the insert is flanked by BsiWI and AsiSI sites. Subsequently,the insert is excised with BsiWI and AsiSI and ligated with the modifiedBAC 27N5 to form BAC 27N5Fos.

The rabbit J region in BAC219D23 was exchanged with the correspondinghuman J region by ET cloning. The human J region was amplified by PCRusing primers SEQ ID NOs 457 and 458 (Table 2).

Unique restriction enzyme sites are inserted in BAC219D23 upstream ofthe D region (A) and upstream of Cμ(B)□. In BAC 27N5Fos restriction siteA is inserted upstream of the linker region and B is inserted insequences homologous to BAC219D23. Following digestion with enzymes Aand B, the fragment containing human J and rabbit D regions is isolatedand ligated with BAC 27N5Fos to create BAC 219D23/27N5Fos.

Chicken heavy chain spacer sequences are amplified from chicken genomicDNA by PCR using primers (SEQ ID NOS 459 and 460, Table 2) specific forchicken heavy chain V pseudogenes (Mansikka et al., J Immunol 145(11),3601-3609 (1990), Reynaud et al., Cell 59(1), 171-183 (1989)).Alternatively, spacer sequences are synthesized chemically.

The PCR products are gel purified, cloned into pTOPO (Invitrogen) andsequenced.

Human heavy chain variable elements are amplified by PCR using primersdesigned according to published sequences in GENBANK (eg Acc. No.NG_001019) or synthesized chemically. The human V elements contain thehuman promoter region, the human leader sequence, the human intronbetween leader and V-coding region, the human V-coding region and thehuman recombination signal sequence. The amplified or synthesizedfragments are flanked by specific restriction endonuclease recognition.Chicken spacer sequences and human V elements are combined in one orseveral large DNA fragment comprising a humanized immunoglobulin locus.The construct is used to generate transgenic animals.

EXAMPLE 10 Construction of a Humanized Immunoglobulin Locus ContainingHuman V Elements and Non-Human Spacer Sequences (without Promoter Regionand RSS)

A BAC library generated with non-human genomic DNA is screened withprobes specific for immunoglobulin and BAC clones containing heavy andlight chain immunoglobulin C, J and D regions are identified. The BACclones are modified to contain restriction enzyme sites. Human heavy andlight chain variable elements are amplified by PCR using primersdesigned according to published sequences in GenBank (eg., Acc. No.NG_001019). Sequences are amplified from genomic DNA or synthesizedchemically. The human V elements contain the human promoter region, thehuman leader sequence, the human intron between leader and V-codingregion, the human V-coding region and the human recombination signalsequence (RSS). The amplified or synthesized fragments have specificrestriction endonuclease recognition sites at the ends. The non-humanspacer sequences are amplified by PCR or synthesized chemically.Non-human spacer sequences and human V elements are combined in one orseveral large DNA fragment comprising a humanized immunoglobulin locus.The construct is used to generate transgenic animals. An example for theconstruction of a humanized V region using chicken spacer sequences isshown in FIG. 10.

EXAMPLE 11 Construction of a Humanized Immunoglobulin Locus ContainingHuman V Elements and Non-Human Spacer Sequences

A BAC library generated with non-human genomic DNA is screened withprobes specific for immunoglobulin and BAC clones containing heavy andlight chain immunoglobulin C, J and D regions are identified. The BACclones are modified to contain restriction enzyme sites. Human heavy andlight chain variable elements are amplified by PCR using primersdesigned according to published sequences in GenBank (eg., Acc No.NG_001019). Sequences are amplified from genomic DNA or synthesizedchemically. The human V elements contain the human V coding region.Non-human spacer sequences are amplified by PCR or synthesizedchemically and contain a recombination signal sequence, a spacersequence, a promoter region, a leader sequence and the intron betweenleader and V coding region. Such non-human spacer sequences are combinedwith human V elements in one or several large DNA fragments and used forthe generation of transgenic animals. An example for the construction ofa humanized V region using mouse or rabbit spacer sequences is shown inFIG. 11.

EXAMPLE 12 Transgenic Rabbits Expressing Humanized Immunoglobulins

Transgenic rabbits were generated as described by Fan et al. (Pathol.Int. 49: 583-594, 1999). Briefly, female rabbits are superovulated usingstandard methods and mated with male rabbits. Pronuclear-stage zygotesare collected from oviduct and placed in an appropriate medium such asDulbecco's phosphate buffered saline supplemented with 20% fetal bovineserum. BAC containing humanized immunoglobulin loci were microinjectedinto the male pronucleus with the aid of a pair of manipulators.Morphological surviving zygotes were transferred to the oviducts ofpseudopregnant rabbits. Pseudopregnancy was induced by the injection ofhuman chorionic gonadotrophin (hCG). Following injection of a humanizedlight chain construct into 4645 pronuclei of fertilized oocytes, 4043oocytes were transferred into 132 recipients. In total, 253 liveoffspring were born, 11 of which were transgenic. Expression of humankappa light chain was detected by ELISA using human-kappa light chainspecific reagents (for example, mouse anti-human Kappa, SouthernBiotech, 9220-01; goat anti-human Kappa, Southern Biotech 2063-08).

A humanized heavy chain construct was injected into 4083 pronuclei offertilized oocytes. 3485 oocytes were transferred into 119 recipientswhich delivered 433 offspring. Analysis by PCR and FISH revealed that 20of these animals were transgenic. Humanized heavy chain in the blood offounder animals was detected by ELISA using antibodies specific forhuman IgM/IgG (for example, rabbit anti-human IgM, Rockland 609-4131;rabbit anti-human IgM, Rockland 609-4631; rabbit anti-human IgG, Pierce31142, rabbit anti-human IgG, Southern Biotech 6145-08; rabbitanti-human IgG, Pierce 31784).

Sandwich-type ELISAs detecting humanized κ, μ and γ chains wereperformed using standard procedures. Briefly, microtiter plates werecoated with capture antibody and incubated with diluted serum samples.Bound human immunoglobulin was detected using a secondary labeledantibody and peroxidase-streptavidin-conjugate (Sigma, S2438).

Double transgenic animals expressing both humanized heavy and lightimmunoglobulin chains were generated by breeding of founder animals.

EXAMPLE 13 Transgenic Mice Expressing Humanized Immunoglobulins

Transgenic mice were generated as described by Nagy et al. (Manipulatingthe Mouse Embryo: A Laboratory Manual; Cold Spring Harbor LaboratoryPress, New York, 2003). Briefly, female mice were superovulated usingstandard methods and mated with male mice. Pronuclear-stage zygotes werecollected from oviduct and placed in a suitable medium such as M2medium. BAC containing humanized immunoglobulin loci were microinjectedinto the male pronucleus with the aid of a pair of manipulators.Morphologically surviving zygotes were transferred to the oviducts ofpseudopregnant female mice. Pseudopregnancy was induced by mating withsterile males. Following injection of a humanized light chain constructinto 1325 pronuclei of fertilized oocytes, 787 oocytes were transferredinto 29 recipients. In total, 55 live offspring were born, 11 of whichwere transgenic.

A humanized heavy chain construct was injected into 1050 pronuclei offertilized oocytes. 650 oocytes were transferred into 25 recipientswhich delivered 64 live offspring. Analysis by PCR revealed that 19 ofthese animals were transgenic.

Double transgenic animals expressing both humanized heavy and lightimmunoglobulin chains are generated by breeding of founder animals.Expression of humanized κ, μ and γ chains was detected by ELISAs usingstandard procedures. Briefly, microtiter plates were coated with captureantibody and incubated with diluted serum samples. Bound humanimmunoglobulin was detected using a secondary labeled antibody andperoxidase-streptavidin-conjugate (Sigma, S2438).

All references cited throughout the specification are hereby expresslyincorporated by reference. While the invention is illustrated byreference to certain embodiments, it is not so limited. One skilled inthe art will recognize that various modifications and variations arepossible without diverting from the essence of the invention. All suchmodifications and variations are specifically included within the scopeherein.

TABLE 1 ID BAC Accession# Start Finish Description 1 38A2 AY386694 12137 Spacer 5′ start-V34 2 38A2 AY386694 2433 9504 Spacer V34-V33 3 38A2AY386694 9798 19384 Spacer V33-V32 4 38A2 AY386694 19690 35164 SpacerV32-V31 5 38A2 AY386694 35447 47669 Spacer V31-V30 6 38A2 AY386694 4797352521 Spacer V30-V29 7 38A2 AY386694 52819 61798 Spacer V29-V28 8 38A2AY386694 62100 74264 Spacer V28-V27 9 38A2 AY386694 74566 79145 SpacerV27-V26 10 38A2 AY386694 79449 84800 Spacer V26-V25 11 38A2 AY38669485103 95717 Spacer V25-V24 12 38A2 AY386694 96009 102226 Spacer V24-V2613 38A2 AY386694 102504 105307 Spacer V23-V22 14 38A2 AY386694 105603107583 Spacer V22-V24 15 38A2 AY386694 107769 118033 Spacer V24-V20 1638A2 AY386694 118334 125546 Spacer V20-V19 17 38A2 AY386694 125849128059 Spacer V19-3′ end 18 225P18 AY386697 1 4333 Spacer 5′ start-V1819 225P18 AY386697 4629 9255 Spacer V18-V17 20 225P18 AY386697 956115841 Spacer V17-V16 21 225P18 AY386697 16135 22502 Spacer V16-V15 22225P18 AY386697 22794 32821 Spacer V15-V14 23 225P18 AY386697 3311837738 Spacer V14-V13 24 225P18 AY386697 38044 44571 Spacer V13-V12 25225P18 AY386697 44865 49447 Spacer V12-V11 26 225P18 AY386697 4974556909 Spacer V11-V10 27 225P18 AY386697 57205 63678 Spacer V10-V9 28225P18 AY386697 63977 71204 Spacer V9-V8 29 225P18 AY386697 71507 76261Spacer V8-V7 30 225P18 AY386697 76560 79012 Spacer V7-V6 31 225P18AY386697 79308 83467 Spacer V6-V5 32 225P18 AY386697 83768 88013 SpacerV5-V4 33 225P18 AY386697 88314 91233 Spacer V4-V3 34 225P18 AY38669791531 95929 Spacer V3-V2 35 225P18 AY386697 96233 100963 Spacer V2-V1 36225P18 AY386697 101262 133721 Spacer V1-D3 37 225P18 AY386697 133752135212 Spacer D3-D1a 38 225P18 AY386697 135237 136922 Spacer D1a-D4 39225P18 AY386697 136947 139446 Spacer D4-3′ end 40 219D23 AY386695 815140612 Spacer V1-D3 41 219D23 AY386695 40643 42102 Spacer D3-D1a 42219D23 AY386695 42127 43812 Spacer D4-D1b 43 219D23 AY386695 43837 48553Spacer D1b-D6 44 219D23 AY386695 48577 48753 Spacer D6-D8 45 219D23AY386695 48769 51181 Spacer D8-D2x 46 219D23 AY386695 51213 55826 SpacerD1c-Df 47 219D23 AY386695 55852 61112 Spacer Df-D1d 48 219D23 AY38669561237 62445 Spacer D1d-D5 49 219D23 AY386695 62471 97024 Spacer D5-DQ5250 219D23 AY386695 97036 97831 Spacer DQ52-J1 51 219D23 AY386695 9787198090 Spacer J1-J2 52 219D23 AY386695 98140 98430 Spacer J2-J3 53 219D23AY386695 98483 98642 Spacer J3-J4 54 219D23 AY386695 98690 98981 SpacerJ4-J5 55 219D23 AY386695 99032 99550 Spacer J5-J6 56 219D23 AY38669599604 106867 1501Spacer J6- IgM exon1 57 219D23 AY386695 107185 107290Spacer IgM exon1- exon2 58 219D23 AY386695 107635 107863 Spacer IgMexon2- exon3 59 219D23 AY386695 108181 108268 Spacer IgM exon3- exon4 60219D23 AY386695 108664 110499 Spacer IgM exon4- exonM1 61 219D23AY386695 110615 110741 Spacer exonM1- exonM2 62 219D23 AY386695 108664137302 Spacer IgM exon4- 3′ end 63 27N5 AY386696 5099 55582 Spacer IgMexon4- IgG exon1 64 27N5 AY386696 55867 56071 Spacer IgG exon1- exon2 6527N5 AY386696 56104 56201 Spacer IgG exon2- exon3 66 27N5 AY386696 5653156623 Spacer IgG exon3- exon4 67 27N5 AY386696 56946 58984 Spacer IgGexon4- exonM1 68 27N5 AY386696 56946 59205 Spacer IgG exon4- exonM2 6927N5 AY386696 56946 69246 Spacer IgG exon4- IgE exon1 70 27N5 AY38669669546 69719 Spacer IgE exon1- exon2 71 27N5 AY386696 70037 70132 SpacerIgE exon2- exon3 72 27N5 AY386696 70453 70532 Spacer IgE exon3- exon4 7327N5 AY386696 70873 73061 Spacer IgE exon4- exonM1 74 27N5 AY38669670873 73292 Spacer IgE exon4- exonM2 75 27N5 AY386696 70873 86059 SpacerIgE exon4- IgA4 exon1 76 27N5 AY386696 86362 86498 Spacer IgA4 exon1-exon2 77 27N5 AY386696 86876 87059 Spacer IgA4 exon2- exon3 78 27N5AY386696 87450 89577 Spacer IgA4 exon3- exonM 79 27N5 AY386696 87450103798 Spacer IgA4 exon3- IgA5b exon1 80 27N5 AY386696 104101 104231Spacer IgA5b exon1- exon2 81 27N5 AY386696 104609 104787 Spacer IgA5bexon2- exon3 82 27N5 AY386696 105182 107227 Spacer IgA5b exon3- exonM 8327N5 AY386696 105182 112077 Spacer IgA5b exon3- exonM 84 27N5 AY386696105182 119223 Spacer IgA5b exon3- IgA1 exon1 85 27N5 AY386696 119511119644 Spacer IgA1 exon1- exon2 86 27N5 AY386696 119984 120162 SpacerIgA1 exon2- exon3 87 27N5 AY386696 120557 122823 Spacer IgA1 exon3-exonM 88 27N5 AY386696 120557 127750 Spacer IgA1 exon3- exonM* 89 27N5AY386696 120557 135838 Spacer IgA1 exon3- IgA2 exon1 90 27N5 AY386696136138 136274 Spacer IgA2 exon1- exon2 91 27N5 AY386696 136652 136831Spacer IgA2 exon2- exon3 92 27N5 AY386696 137229 139433 Spacer IgA2exon3- exonM 93 27N5 AY386696 137229 146676 Spacer IgA2 exon3- 3′ end 94Fos15B AY386698 1 828 Spacer 5′ start-IgA exonM 95 Fos15B AY386698 10433596 Spacer IgA exonM- end contig1 96 Fos15B AY386698 1 3596 Spacer 5′start-end contig1 97 Fos15B AY386698 7404 7541 Spacer IgA exon1- exon298 Fos15B AY386698 7908 8086 Spacer IgA exon2- exon3 99 Fos15B AY3866988481 10538 Spacer IgA exon3- exonM 100 Fos15B AY386698 8481 13140 SpacerIgA exon3- end contig2 101 Fos15B AY386698 8481 15871 Spacer IgA exon3-1,2 hs 3′ enh 102 Fos15B AY386698 8481 21447 Spacer IgA exon3- 4hs enh103 Fos15B AY386698 21484 33297 Spacer 4hs enh-end contig4 104 Fos15BAY386698 16633 33297 Spacer 1,2hs 3′ enh- end contig4 105 179L1 AY4958271 124285 Spacer 5′ end-enh 106 179L1 AY495827 125411 131350 Enhancer-C□107 179L1 AY495827 131664 134637 Spacer C□-J5 108 179L1 AY495827 134684134915 Spacer J5-J4 109 179L1 AY495827 134952 135196 Spacer J4-J3 110179L1 AY495827 135241 135485 Spacer J3-J2 111 179L1 AY495827 135525135863 Spacer J2-J1 112 179L1 AY495827 135897 155257 Spacer J1-V1 113179L1 AY495827 155572 170621 Spacer V1-V2 114 179L1 AY495827 170936173443 Spacer V2-V3 115 179L1 AY495827 173752 177227 Spacer V3-V4 116179L1 AY495827 177536 185356 Spacer V4-V5 117 179L1 AY495827 185664200758 Spacer V5-V6 118 179L1 AY495827 201064 203580 Spacer V6-V7 119179L1 AY495827 203886 205144 Spacer V7-3′ end 120 215M22 AY495826 112829 Spacer 5′ end-V6 121 215M22 AY495826 13136 15653 Spacer V6-V7 122215M22 AY495826 15957 22241 Spacer V7-V8 123 215M22 AY495826 22551 32876Spacer V8-V9 124 215M22 AY495826 33188 38276 Spacer V9-V10 125 215M22AY495826 38582 41476 Spacer V10-V11 126 215M22 AY495826 41780 47827Spacer V11-V12 127 215M22 AY495826 48133 48547 Spacer V12-V13 128 215M22AY495826 48841 51408 Spacer V13-V14 129 215M22 AY495826 51638 55438Spacer V14-V15 130 215M22 AY495826 55745 67437 Spacer V15-V16 131 215M22AY495826 67743 77805 Spacer V16-V17 132 215M22 AY495826 78120 80628Spacer V17-V18 133 215M22 AY495826 80937 84009 Spacer V18-V19 134 215M22AY495826 84315 87339 Spacer V19-V20 135 215M22 AY495826 87648 89399Spacer V20-V21 136 215M22 AY495826 89711 95414 Spacer V21-V22 137 215M22AY495826 95720 106650 Spacer V22-V23 138 215M22 AY495826 106956 110940Spacer V23-V24 139 215M22 AY495826 111246 117877 Spacer V24-V25 140215M22 AY495826 118183 122396 Spacer V25-V26 141 215M22 AY495826 122706126496 Spacer V26-V27 142 215M22 AY495826 126802 133358 Spacer V27-V28143 196O2 AY495828 37134 48826 Spacer V15-V16 144 196O2 AY495828 4903259195 Spacer V16-V17 145 196O2 AY495828 115057 125885 Spacer V28-V29 146196O2 AY495828 126195 130012 Spacer V29-V30 147 196O2 AY495828 130318136966 Spacer V30-V31 148 196O2 AY495828 137272 144512 Spacer V31-V32149 196O2 AY495828 144819 148617 Spacer V32-V33 150 196O2 AY495828148923 155402 Spacer V33-V34 151 196O2 AY495828 155714 171415 SpacerV34-V35 152 196O2 AY495828 171572 177676 Spacer V35-V36 153 196O2AY495828 177979 178083 Spacer V36-3′ end 154 CLC* NA 1 443 Spacer 5′end- pV28** 155 CLC* NA 486 1203 Spacer pV28- Pv27** 156 CLC* NA 15281635 Spacer pV27- pV26** 157 CLC* NA 1818 2242 Spacer pV26- pV25** 158CLC* NA 2585 2676 Spacer pV25- pV24** 159 CLC* NA 2781 3327 Spacer pV24-pV23** 160 CLC* NA 3464 3659 Spacer pV23- pV22** 161 CLC* NA 3985 4241Spacer pV22- pV21** 162 CLC* NA 4578 4994 Spacer pV21- pV20** 163 CLC*NA 5366 5425 Spacer pV20- pV19** 164 CLC* NA 5749 5842 Spacer pV19-pV18** 165 CLC* NA 6034 7043 Spacer pV18- pV17** 166 CLC* NA 7266 7493Spacer pV17- pV16** 167 CLC* NA 7625 7625 Spacer pV16- pV15** 168 CLC*NA 7988 8758 Spacer pV15- pV14** 169 CLC* NA 9100 9410 Spacer pV14-pV13** 170 CLC* NA 9787 10057 Spacer pV13- pV12** 171 CLC* NA 1044111022 Spacer pV12- pV11** 172 CLC* NA 11380 11911 Spacer pV11- pV10**173 CLC* NA 12162 12349 Spacer pV10-pV9** 174 CLC* NA 12691 13357 SpacerpV9-pV8** 175 CLC* NA 13708 13882 Spacer pV8-pV7** 176 CLC* NA 1422914406 Spacer pV7-pV6** 177 CLC* NA 14599 15338 Spacer pV6-pV5** 178 CLC*NA 15613 16578 Spacer pV5-pV4** 179 CLC* NA 16916 18219 Spacer pV4-pV3**180 CLC* NA 18439 18879 Spacer pV3-pV2** 181 CLC* NA 19248 19343 SpacerpV2-pV1** 182 CLC* NA 19609 22208 Spacer pV1-V** 183 CLC* NA 22506 24313Spacer V-J 184 CLC* NA 24350 26088 Spacer J-C□ 185 CLC* NA 26402 36259Spacer C-3' end *CLC—Chicken light chain locus SEQ ID 184, FIG. 9**pV—pseudo V gene (not functional) Comments: BAC sequences submitted toGenBank were modified by deletion of vector sequences at the 5' and 3'end as follows: BAC Accession# Removed from 5′ end Removed from 3′ end38A2 AY386694 1-125 1281285-128225 219D23 AY386695 1-54  137357-137389Fos15B* AY3866968 1-97  33395-33427 179L1 AY495827 0  205145-205968196O2 AY495828 1-32  178117-178171 *In addition contigs in GenBank areseparated by 50 nt. In the Fos15B sequence submitted with theprovisional application contigs were separated by 10 nt.

TABLE 2 ID Region Sequence 193 V_(H)1 5′CGCGGATCCGAGACTGGGCTGCGCTG3′ 194V_(H)1 5′CGCAAGCTTGAAATAGGTGGCCGTGTC3′ 195 J_(H)5′CGCGGATCCAGGCACCCTGGTCACCG3′ 196 J_(H) 5′CGCAAGCTTGTGACCAGGGTGCCCTG3′197 Cγ 5′CGCGGATCCCTGGAGCCGAAGGTCTAC3′ 198 Cγ5′CGCAAGCTTGAGATGGACTTCTGCGTG3′ 199 3'Enh5′CGCGGATCCCAGAGTGGGTCTGTGACA3′ 200 3'Enh5′CGCAAGCTTACAGGCGCATGCAAATGC3′ 201 Vκ 5′CGCGGATCCGAGGCACAGTCACCATC3′202 Vκ 5′CGCAAGCTTACAGTAGTAAGTGGCAGC3′ 203 Jκ5′CGCGGATCCGGAGGGACCGAGGTGGT3′ 204 Jκ 5′CGCAAGCTTACCATGGTCCCTGAGCC3′ 205C□ 5′CGCGGATCCCCTCAGGTGATCCAGTTG3′ 206 C□5′CGCAAGCTTCTATTGAAGCTCTGGACG3′ 207 K 3'Enh5′CGCGGATCCGTGACTGGCCCAAGAAG3′ 208 K 3'Enh5′CGCAAGCTTATACAACCTTGGCCAGG3′ 209 C□5′AAACAGCTTTTCACACCTCCCCTTTCTCTCTTTGCTCCCCTGGGCCCTCAGGGAGTGCATCCGCCCCAACCCTTTTCC3′ 210 C□5′CAGGGTTAGTTTGCATGCACACACACACAGCGCCTGGTCACCCAGAGGGGTCAGTAGCAGGTGCCAGCTGTGTCGGACATG3′ 211 C□5′GGTCAGGGGTCCTCCAGGGCAGGGGTCACATTGTGCCCCTTCTCTTGCAGCCTCCACCAAGGGCCCATCGGTC3′ 212 Cγ5′CACAGCTGCGGCGTGGGGGGGAGGGAGAGGGCAGCTCGCCGGCACAGCGCTCATTTACCCGGAGACAGGGAGAGGCTCTTC3′ 213 J_(H)5′GTGTTATAAAGGGAGACTGAGGGGGCAGAGGCTGTGCTACTGGTACCTGGCTGAATACTTCCAGCACTGGGGCCAGG3′ 214 J_(H)5′GGCCACAGAAAAGAGGAGAGAATGAAGGCCCCGGAGAGGCCGTTCCTACCTGAGGAGACGGTGACCGTGGTCCCT TG-3′ 215 Genta5′CCAGGCCGGCCTGGAGTTGTAGATCCTCTACG3′ 216 Genta5′CCAGGCGCGCCAAGATGCGTGATCTGATCC3′ 217 Linker5′GGCCGCGGCCGGCCATCGATGGCGCGCCTTCGAAACGCGTA3′ 218 Linker5′AGCTTACGCGTTTCGAAGGCGCGCCATCGATGGCCGGCCGC3′ 219 pBB11.15′ATTCCCAAGCTTTTAATTAAGACGTCAGCTTCCTTAGCTCCTG3′ 220 pBB11.15′ATTCGCGGATCCACGCGTTTCGTTCCCAAAGGCGCGCCTAGCG ATGAGCTCGGAC3′ 221 Neo5′GCAGGCATGCAAAGCTTATTACACCAGTGTCAGTAAGCG3′ 222 Neo5′GGTACCCGGGGATCCTCAGAAGAACTCGTCAAGAAGGCG3′ 223 pBB11.25′AAATTCCCTTAATTAAGACGTCAGCTTCCTTAGCTCCTG3′ 224 pBB11.25′GAAACCGGGGACGCGTTACCGTTCGTATAATGTATGCTATACGAAGTTATGCGGCCGCTAGCGATGAGCTCGGAC3′ 225 CA5′TTCTCTGTTTTTGTCCGTGGAATGAACAATGGAAGTCCGAGCTCATCGCTAAGGGCACCAATAACTGC3′ 226 CA5′CACAGGAGAGAAACAGGACCTAGAGGATGAGGAAGTCCCTGTAGGCTTCCTACCGTTCGTATAATGTATGCTATACGAAGTTATTACCTGT GACGGAAGATC-3′ 227V_(H)3-9 5′ATAGAGAGATTGAGTGTG3′ 228 V_(H)3-9 5′TCCTGTCTTCCTGCAG3′ 229V_(H)3-11 5′AGAGACATTGAGTGGAC3′ 230 V_(H)3-11 5′AGGGAGGTTTGTGTC3′ 231V_(H)3-13 5′ACTAGAGATATTGAGTGTG3′ 232 V_(H)3-13 5′AGGCATTCTGCAGGG3′ 233V_(H)3-15 5′ACTAGAGAGATTAAGTGTG3′ 234 V_(H)3-15 5′TCACACTGACCTCCC3′ 235V_(H)3-20 5′TCATGGATCAATAGAGATG3′ 236 V_(H)3-20 5′TGCAGGGACGTTTGTG3′ 237V_(H)3-23 5′AGAAAAATTGAGTGTGAA3′ 238 V_(H)3-23 5′GTGTCTGGGCTCACAA3′ 239V_(H)3-30 5′AGAGAGACTGAGTGTG3′ 240 V_(H)3-30 5′TGCAGGGAGGTTTGTG3′ 241V_(H)3-43 5′TGAGTGTGAGTGAACATG3′ 242 V_(H)3-43 5′ACCAGCTCTTAACCTTC3′ 243V_(H)3-64 5′TGAGTGTGAGTGGAC3′ 244 V_(H)3-64 5′TGACGCTGATCAGTG3′ 245V_(H)3-66 5′TCTGACCAATGTCTCTG3′ 246 V_(H)3-66 5′AGGTTTGTGTCTGGGC3′ 247V_(H)3-72 5′ACAAGGTGATTTATGGAG3′ 248 V_(H)3-72 5′AGGTTTGTGTCCGGG3′ 249V_(H)3-9 5′TTGGCGCGCC TGTCGTCTGTGTTTGCAG GTGTCC3′ 250 V_(H)3-95′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCAGCCTGAGGGCCCCTCACTGTGTCATCTTTTGCAC3′ 251 V_(H)3-115′TTGGCGCGCC TGTCGTCTGTGTTTGCAG GTGTCC3 252 V_(H)3-115′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA GCCTGAGGGCCCCTCACTGTGTCTCTCG3′ 253V_(H)3-13 5′TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3′ 254 V_(H)3-135′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA GCCTGAGGGCCCCTCACTGTGTCTCTTG3′ 255V_(H)3-15 5′TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3′ 256 V_(H)3-155′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA GCCTGAGGGCCCCTCACTGTGTCTGTGG3′ 257V_(H)3-20 5′TTGGCGCGCC TGTCGTCTGTGTTTGCAGGTGTC3′ 258 V_(H)3-205′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTC AGCCTGAGGGCCCCTCACTGTGTCTCTC3′ 259V_(H)3-23 5′TTGGCGCGCCTGTCGTCTGTGTTTGCAG GTGTCCAGTGTG3′ 260 V_(H)3-235′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCAGCCTGA GGGCCCCTCACTGTGTCTTTC3′ 261V_(H)3-30 5′TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCCAGTGTC3′ 262 V_(H)3-305′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCAGCCTG AGGGCCCCTCACTGTGTCTTTCG3′ 263V_(H)3-43 5′TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3′ 264 V_(H)3-435′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCAGCCTGAGGGCCCCTCACTGTGTCTCTTTTGCAC3′ 265 V_(H)3-645′TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3′ 266 V_(H)3-645′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA GCCTGAGGGCCCCTCACTGTGTCTCTCGCAC3′267 V_(H)3-66 5′TTGGCGCGCCTGTCGTCTGTGTTTGCAGGTGTCC3′ 268 V_(H)3-665′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA GCCTGAGGGCCCCTCACTGTGTCTCCG3′ 269V_(H)3-72 5′TTGGCGCGCCTGTCGTCTGTGTTTGCAG GTTTCC3′ 270 V_(H)3-725′TTGCACGCGTGCAGGGAGGTTTGTGTCTGGGCTCA GCCTGAGGGCCCCTCACTGTGTCTCTAGCAC3′271 V1-2 5′TTGGCGCGCCAGGGGAGTGCGGCTCCAC3′ 272 V1-25′TTGCACGCGT TGGTCAGGACACTGTCACTCAC3′ 273 V2-35′TTGGCGCGCCAGGGGCGCGCGGCTCCAC3′ 274 V2-35′TTGCACGCGTTGATCACGAAACTGTCACTCACACTCTC3′ 275 V3-45′TTGGCGCGCCAGGGGCGCGCGGCTCCAC3′ 276 V3-45′TTGCACGCGTTCTGTTGGTCTCTTCTTCTCTTGCTATAAC3′ 277 V4-55′TTGGCGCGCCAGGGGAGTGCGGCTCCAC3′ 278 V4-55′TTGCACGCGTTGGTCAAGACACTGTCACTCAC3 279 V5-65′TTGGCGCGCCAGGGACGCACGGCTCCAC3′ 280 V5-65′TTGCACGCGTTGGTCAGGAAGCTGTCACTCAC3′ 281 V6-75′TTGGCGCGCCAGGGATGCGCGGCTCCAG3′ 282 V6-75′TTGCACGCGTTGGTCAGGACACTGTCACTGACAC3′ 283 V7-85′TT GGCGCGCCAGGGGAGTGCGGCTCCAC3′ 284 V7-85′TTGCACGCGTTGGTCAGGAAGCTGTCACTCACTCTC3′ 285 V21-225′TTGGCGCGCCGGGGCCCGCGGCTCCAC3′ 286 V21-225′TTGCACGCGTTGGTCAGGAAGCTGTCAC3′ 287 V22-235′TTGGCGCGCCAGGGACGTGAGGCTCTAC3′ 288 V22-235′TTGCACGCGTTGGTCAGGGCACTGTCAC3′ 289 Linker5′GGCCGCGGCCGGCCATCGATGGCGCGCC TTCGAAACGCGTA3′ 290 Linker3′CGCCGGCCGGTAGCTACCGCGCGGAAGCTT TGCGCATTCGA5′ 291 Linker5′CGG CCG GCC ATC GAT GGC GCG CCT TCG AAA CGC GTG GTA C3′ 292  Linker3′TCG AGC CGG CCG GTA GCT ACC GCG CGG AAG CTT TGC GCA C5′ 293 Genta5′CCAGGCCGGCCTGGAGTTGTAGATCCTCTACG3′ 294 Genta5′CCAGGCGCGCCAAGATGCGTGATCTGATCC-3′ 295 Neo5′CCAGGCCGGCCATTACACCAGTGTCAGTAAGCG3′ 296 Neo5′CCAGGCGCGCCTCAGAAGAACTCGTCAAGAAGGCG3′ 297 Linker5′GAT CCG GCC GGC CAT CGA TGG CGC GCC TTC GAA ACG CGTTAG GGA TAA CAG GGT AAT A3′ 298 Linker3′GCC GGC CGG TAG CTA CCG CGC GGA AGC TTT GCG CAA TCCCTA TTG TC CCA TTA TCGA5′ 299 Neo5′ATCTGCACTCAGTGCGTCTTGAGCGCCCCCTGGTAGAGCCGCGCGACCCT GGCGCGCC ATTACACCAGTGTCAGTAAGCG3′ 300 Neo5′AAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGA GGCCGGCC TCAGAAGAACTCGTCAAGAAGGCG3′ 301 Cκ Km35′GATGTCCACTGGTACCTAAGCCTCGCCCTCTGTGCTTCTTCCCTCCTCAGGAACTGTGGCTGCACCATCTGTCTTC3′ 302 Cκ Km35′GAGGCTGGGCCTCAGGGTCGCTGGCGGTGCCCTGGCAGGCGTCTCGCTCTAACACTCTCCCCTGTTGAAGCTCTTTGTG3 303 Neo5′CTTTCTCTGTCCTTCCTGTGCGACGGTTACGCCGCTCCATGAGCTTATCGTAACTATAACGGTCCTAAGGTAGCGATGGACAGCAAGCGAA CCGGA3′ 304 Neo5′GGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCATCGTGTCAGAAGAACTCGTCAAGAAG3′ 305 Zeo5′CCCCCCCCGCCACTTCTCTTCTGTTTCGTTTAAGTTCTACACTGACATACTAGGGATAACAGGGTAATAACGTTTACAATTTCGCCTGATG3 306 Zeo5′AGTGGGTAGGCCTGGCGGCCGCCTGGCCGTCGACATTTAGGTGACACTATAGAAGGATCCTAGCACGTGTCAGTCCTGCT3′ 307 Genta 5′TTACGCCAAGCTATTTAGGTGACACTATAGAATACTCAAGCTTTGATTGCTAACTATAACGGTCCTAAGGTAGCGATGAAGGCACGAACCC AGTTG3′ 308 Genta 5′GCGGAATTCTATGTCTAGTGGAGGGTGAAGCTGGTGATTATAGAGTGAAAATTACCCTGTTATCCCTATCGGCTTGAACGAATTGTTAG3′ 309 VJ5′CATAAATATACTGTCTTCCAGGATCTTAGAGCTCACCTAAGGAAACAAGAGTTCATTTGAAGTTTTTAAAGTG3′ 310 VJ5′ACTCCAGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCCTTTGATCTCCACCTTGGTC3′ 311 Genta 5′GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTGGAGTTGTAGATCCTCTACG3′ 312 Genta  5′AAAACAAACCAATCAGGCAGAAACGGTGAGGAATCAGTGAAACGGCCACTTACGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAGATGCGTGATCTGATCC3′ 313 FRT 5′TTATGCTGCATCCAGTTTGC3′ 314FRT 5′AAAACAAACCAATCAGGCAG3′ 315 FRT 5′TGTGACATCCAGATGAC3′ 316 FRT5′AAAACAAACCAATCAGGCAG3′ 317 Genta5′GGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCATCGTGGTGCCAGGGCGTGCCCTTGGGCTGGGGGCGCGATAACTTCGTATAGCATACATTATACGAAGTTATCGATCGTGGAGTTGTAGATCC TCTACG3′ 318 Genta5′TTACGCCAAGCTATTTAGGTGACACTATAGAATACTCAAGCTTTGATTGCAAGATGCGTGATCTGATCCT3′ 319 Linker5′CGGGATCCGCGCGTACGGAAGTTCCTATACCTTTTGAAGAATAGGAACTTCGGAATAGGAACTTCATTACACCAGTGTCAGTAAGCG3′ 320 Linker5′GGGAAGCTTCGCGCGATCGCCGCTTTCGCAAAGGCGCGCCTCAG AAGAACTCGTCAAGAAGGCG3′321 Genta 5′GGCGGCCGCCTGGCCGTCGACATTTAGGTGACACTATAGAAGGATCCGCGTGGAGTTGTAGATCCTCTACG3′ 322 Genta5′AACTCAGTAAGGAAAAGGACTGGGAAAGTGCACTTACATTTGATCTCCAGGCGCGCCAAGATGCGTGATCTGATCC3′ 323 Neo5′GGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCATCGTGGTGCCAGGGCGTGCCCTTGGGCTGGGGGCGCGGAAGTTCCTATTCCGAAGTTCCTATTCTTCAAAAGGTATAGGAACTTCCGTACGA TTACACCAGTGTCAGTAAGCG3′324 Neo 5′GGACTGATGGGAAAATAGAGGAGAAAATTGACCAGAGGAAGTGCAGATGGTCAGAAGAACTCGTCAAGAAGGCG3′ 325 RSS5′AACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTTCCACAGTGATACAAGCCC3′ 326 RSS5′TGCCGGCCACGATGCGTCCGGCGTAGAGGATCTACAACTCCAGGCGCGCCTGGTCATGTCAGTGCTGCTGC3′ 327 Genta5′CTCCTTTCCTCCTCCTTGGTGGCAGCAGCACTGACATGACCAGGCGCGCC TGGAGTTGTAGATCCTCTACG3′ 328 Genta5′TGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGGGGATCCCGTACGAAGATGCGTGATCTGATCC3′ 329 Kana5′GGCGGCCGCCTGGCCGTCGACATTTAGGTGACACTATAGAAGGATCCGCGACCCTGTTATCCCTAGATTTAAATGATATCGG3′ 330 Kana5′AACTTTCTCCTACAGATCCCAGATAACCATGAATTTATTACACCATCTTGGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAGTTGGTGATTTTGAACTTTTGCTTTGCC3′ 331 Amp5′GGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCATCGTGGTGCCAGGGCGTGCCCTTGGGCTGGGGGCGCGGCGATCGCGAAGTTCCTATTCCGAAGTTCCTATTCTTCAAAAGGTATAGGAACTTC TACGGGGTCTGACGCTCAG3′332 Amp 5′GAATTCAGAGCTCAATGAGTTGCCTTGTTCAGAGCTCTATTTTCACTTGACGTACGACAGACAAGCTGTGACCGTC3′ 333 J Region5′GAGTTAGGCCTCAGAGCTGAGGCAGGGCTCGGTTCCCCTTGGGTGAGAAGGGTTTCTGTTCAGCAAGAC3′ 334 J Region5′TGGCCAATTAGAGCAAAATTTCAGACAGTAATAGGAAAAAGGTACTTACGTTTAATCTCCAGTCGTGTC3′ 335 O2 5′TCAGTACTGACTGGAAC3′ 336 O25′CCAATGACTTTCAAAACC3′ 337 L8 5′CCGTACAGCCTGGCTC3′ 338 L85′AACACCATCAGAGTGTGC 339 L4 5′ATGATTAATTGTGTGGACC3′ 340 L45′AGGTGATCTCATATCCTC3′ 341 A30 5′CTCAGTACTGCTTTACTG3′ 342 A305′TGACTTCATGTCCCCTTC3′ 343 L11  5′ACATGATTAATTGTGTGGACC3′ 344 L115′GGTGCAGAGGTGACTTCG3′ 345 L1 5′CTCAGTACTGCTTTACTATTC3′ 346 L15′GAGGAACACTCTCAGCTG3′ 347 L5 5′CAGGGAACTTCTCTTACAG3′ 348 L55′GAATTAGGGTGCAGAGGC3′ 349 L15 5′TACTATTCAGGGAAATTC3′ 350 L155′TGTCTGTGAAGTTGGTG3′ 351 O8 5′TGGCTCTTGATGGAAGC3′ 352 O85′ACTTCAAAGTGTGACTGC3′ 353 L19 5′AGGGAACTTCTCTTACAGC3′ 354 L195′AATTAGGGTGCAGAGGCG3′ 355 L12 5′GAAGTCTTCCTATAATATGATC3′ 356 L125′TGGCTGCATCTGAGGACC3′ 357 A20 5′GCCACTAATGCCTGGCAC3′ 358 A205′CTGCTGTCAGCAGAGGGC3′ 359 O4 5′CTTCTTATAACATGATGG3′ 360 O45′AAACGCTCTGAGCAGC3′ 361 L14 5′CTCAGTACTGCTTTACTG3′ 362 L145′GAGGAACAATCTCAGCCG3′ 363 L23 5′AGCCAGGCTGTACGGAAC3′ 364 L235CCCAGCCTCACACATCTC3′ 365 L9 5′TGGCCCTTCAGGGAAG3′ 366 L95′ACCATCAGAGTGTGGTTG3′ 367 A4 5′CCAGTGTAGCCATTAATG3′ 368 A45′TACCAAAACTTCCCAGGG3′ 369 L24 5′GGGAAATTCTCTTACTAC3 370 L245′CCCCCTCTACCAATAC3′ 371 O6 5′CCATTCAGGGAAGTCTTC3′ 372 O65′TGAGTCTGAGAAGTGTTG3′ 373 L22 5′GGAATTTTCTTAGCCCAC3′ 374 L225′ATGTTCAGGCTTGTAACC3′ 375 A9 5′TCATCTTACAAATAGTTG3′ 376 A95′TCTGACCATTCCTGC3′ 377 A25 5′GGGAAATCATCTTATAAATAG3′ 378 A255′TGCAGATGAGACTTCTGG3′ 379 A15 5′ATTCAGGAAAGTCCTCTC3′ 380 A155′CAGTGACCTTCAGAGTG3′ 381 O9 5′ATTCAGGAAAGTCCTCTC3′ 382 O95′CAGTGACCTTCAGAGTG3′ 383 O25′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC 384 O25′CGCACGCGTGTTTGATCTCCACCTTGGTCCCTCCGCCGAAAGTGA GAGGGGTACTGTAACTCTGTTG3′385 L8 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC 386 L85′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTAAG3′ 387L4 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGCCATC 388 L45′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTAAAC3′ 389 A30 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGGTGTGACATC3′ 7 390  A305′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTATGC3′ 391 L11 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGCCATC3′ 392  L115′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAATTGTAATC3′ 393L1 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC3′ 394 L15′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTATAC3′ 395L5 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTTCCAGATGCGACATC3′ 396 L55′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGAAACTGTTAG3′ 397L15 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC 398 L155′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTATAC3′ 399O8 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC 400 O85′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGAGATTATCATAC3′ 401L19 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTTCCAGATGCGACATC3′ 402 L195′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGAAACTGTTAG3′ 403L12 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAAATGTGACATC3′ 404 L125′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGAATAACTATTATAC3′ 405A20 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGATACCAGATGTGACATCC3′ 406 A205′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGGCACTGTTATAC3′ 407O4 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGACATC3′ 408 O45′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGGCATTGTAAG3′ 409L14 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTAACATCC3′ 410 L145′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATTATGC3′ 411L23 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGCCATC3′ 412 L235′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGGTACTATAATAC3′ 413L9 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGCCATC3′ 414 L95′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTATAATAC3′ 415A4 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGATACCAGATGTGACATCC3′ 416 A45′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGGCACTGTTATAC3′ 417L24 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATGTGTCATC3′ 418 L245′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGAAACTATAATAC3′ 419O6 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGGACCAGAAGTGACATC3′ 420 O65′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAATTTTTATAC3′ 421L22 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGTCAGATTTGACATCC3′ 422 L225′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTAACTGAAGTC3′ 423A9 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGAGTCAGATGTGATTTCC3′ 424 A95′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GATGGCTGCTGTAAG3′ 425A25 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGAGTCAGATGTGATTTC C3′ 426 A255′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GATGGCTGCTGTAAG3′ 427A15 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATATGACATGC3′ 428 A155′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTCACTTTTATAC3′ 429O9 5′TTGGCGCGCCTTCTGTTTCCCTTCTCAGGTGCCAGATATGACATGC3′ 430 O95′CGCACGCGTCCTGGGGGGTTTTTGTTAGGGCTTGTATCACTGTGG GAGGGTCACTTTTATAC3′ 431V7-8 5′TTGGCGCGCC GGAGGAAACAGAAACACAG3′ 432 V7-85′CGCACGCGT CAGCTGCTCGTCCTGGG3′ 433 V11-105′TTGGCGCGCC GGAGGGAAACAGAAACAC3′ 434 V11-105′CGCACGCGTAGCTGCTCCTCCTGGG3′ 435 V15-145′TTGGCGCGCC GAAGGGAAACAGAAACACAG3′ 436 V15-145′CGCACGCGTAGCTGCTCCTCCTGGG3′ 437 V18-175′TTGGCGCGCC GAGGGAAACAGAAACAC3′ 438 V18-175′CGCACGCGTCAGCTGCTGCTCCTGGG3′ 439 V19-185′TTGGCGCGCC GAAGGGAAACAGAAACACAG3′ 440 V19-185′CGCACGCGT AGCTGCTCCTCCTGGG3′ 441 V20-195′TTGGCGCGCC GAGGAGGGAAACAGAAACAC3′ 442 V20-195′CGCACGCGTCAGCTGCCCCTCCTGGG3′ 443 V21-205′TTGGCGCGCC GGAGGAAACAGAAACACAG3′ 444 V21-205′CGCACGCGTCCCTAGCTGCTCCTGGG3′ 445 V24-235′TTGGCGCGCC GGAGGGAAACAGACACAC3′ 446 V24-235′CGCACGCGTCAGCTGCTCCTCCTGGC3′ 447 V26-255′TTGGCGCGCC GAAGGGAAAGAGAAACACAG3′ 448 V26-255′CGCACGCGTAGCTGCTCCTCCTGGG3′ 449 V27-265′TTGGCGCGCC GGAGGGAAACAGAAACAC3′ 450 V27-265′CGCACGCGTCCCAGCTGCTCCTGGG3′ 451 Genta5′AGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCCGGCCTGGAGTTGTAGATCCTCTACG3′ 452 Genta5′AAAACAAACCAATCAGGCAGAAACGGTGAGGAATCAGTGAAACGGCCACTTACGGCGCGCCAAGATGCGTGATCTGATCC3′ 453 Hygro5′CGTTGGACCAGTTTACAATCCCACCTGCCATCTAAGAAAGCTGGTCTCATATAACTTCGTATAATGTATGCTATACGAACGGTAACGCGTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTCAGAGCAGATTGTACTG3′ 454 Hygro5′GGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTAAGATGCGTGATCTGATCC3′ 4555′ACTGCACCTCAGCGTCCCCCTGCCCATGTCAGGGCCGATGAAGGGCACAGCGTACGATTACACCAGTGTCAGTAAGCG3′ 4565′TGTAATACGACTCACTATAGGGCGAATTGAGCTCGGTACCCGGGGATCCTGCGATCGCTCAGAAGAACTCGTCAAGAAGGCG3′ 457 J_(H)5′GTGTTATAAAGGGAGACTGAGGGAGGCAGAGGCTGTGCTACTGGTACCTGGCTGAATACTTCCAGCACTGGGGCCAGG3′ 458 J_(H)5′GGCCACAGAAAAGAGGAGAGAATGAAGGCCCCGGAGAGGCCGTTCCTACCTGAGGAGACGGTGACCGTGGTCCCTTG3′ 459 Spacer5′AACAACCTCAGGGCTGAGGACACC3′ 460 Spacer5′CTGCCCGTTGTCCCTCGAGATGGTGGCACGGCC3′

What is claimed is:
 1. A B cell from a transgenic rabbit, wherein saidtransgenic rabbit comprises a humanized immunoglobulin (Ig) locuscomprising multiple Ig gene segments, present in a transgenic vectorwherein: (a) at least one of said gene segments is a human Ig genesegment comprising two or more identical or different units consistingof, from 5′ to 3′ direction, a 5′ nucleotide sequence, a humanimmunoglobulin heavy or light chain V gene segment, and a 3′ nucleotidesequence, wherein said 5′ nucleotide sequence and said 3′ nucleotidesequence have the nucleotide sequence of SEQ ID NO: 35; (b) said genesegments are juxtaposed in an unrearranged, partially rearranged orfully rearranged configuration, and (c) said humanized Ig locus iscapable of undergoing gene rearrangement, if necessary, and geneconversion and/or hypermutation, and producing a repertoire of humanizedimmunoglobulins in said transgenic rabbit.
 2. The B cell of claim 1,wherein said transgenic rabbit preserves an essentially intactendogenous regulatory and antibody production machinery.
 3. The B cellof claim 1 or claim 2, wherein said human Ig heavy chain V gene segmentis a member of the VH3, VH1, VH5, or VH4 family.